Bacillus subtilis strains improving animal performance parameters

ABSTRACT

The present invention provides a  Bacillus subtilis  strain selected from the group consisting of a) the strain deposited as DSM32324,b) the strain deposited as DSM32325, and c) a mutant strain of (a) or (b) which has sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and has inhibitory activity against  E. coli  and Clostridium perfringens. The invention further relates to  Bacillus  compositions comprising at least one  Bacillus subtilis  strain of the invention, preferably the  Bacillus subtilis  strain DSM32324 and/or the  Bacillus subtilis  strain DSM32325, as Direct Fed Microbial (DFM), premix, animal feed additive or animal feed. The invention provides a method of improving one or more animal performance parameters selected from the group consisting of i) increased weight gain (WG), ii) lower feed conversion ratio (FCR), iii) lower necrotic enteritis lesion scoring, iv) lower necrotic enteritis frequency, v) lower necrotic enteritis mortality, vi) increased European Production Efficacy Factor (EPEF), and vii) lower mortality, by feeding a strain or a composition according to the invention to an animal.

FIELD OF THE INVENTION

The present invention provides a Bacillus subtilis strain selected from the group consisting of a) the strain deposited as DSM32324, b) the strain deposited as DSM32325, and c) a mutant strain of (a) or (b) which has sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and has inhibitory activity against Escherichia coli and Clostridium perfringens.

The invention further relates to Bacillus compositions comprising at least one Bacillus subtilis strain of the invention, preferably the Bacillus subtilis strain DSM32324 and/or the Bacillus subtilis strain DSM32325, as Direct Fed Microbial (DFM), premix, animal feed additive or animal feed.

The invention provides a method of improving one or more animal performance parameters selected from the group consisting of i) increased weight gain (WG), ii) lower feed conversion ratio (FCR), iii) lower necrotic enteritis lesion scoring, iv) lower necrotic enteritis frequency, v) lower necrotic enteritis mortality, vi) increased European Production Efficacy Factor (EPEF), and vii) lower mortality, by feeding a strain or a composition according to the invention to an animal.

BACKGROUND OF THE INVENTION

The phase-out of antibiotic growth promoters in the European Union in 2006 has resulted in an increased need for cost-effective feed additives with high efficacy and susceptibility to inhibitorys of human and veterinary importance.

Bacillus-based probiotic feed additives are known for their positive effects on health and production in pigs and poultry. These products are relevant for the feed industry because spores are heat stable and can survive the pelletizing process at temperatures up to 90-95° C. The endospore-forming bacteria Bacillus subtilis and Bacillus licheniformis are Generally Regarded as Safe (GRAS) by the U.S. Food and Drug Administration (FDA) and acceptable for inclusion in an animal diet or water by the Association of American Feed Control Officials (AAFCO).

WO2013/153159 describes a method for selecting a Bacillus strain having antibiotic sensitivity, inhibitory activity against E.coli and Clostridium perfringens, and high sporulation.

Many of the isolates screened showed undesirable antibiotic resistance above breakpoints defined by the European Food Safety Authority (EFSA) and were discarded due to safety concerns. Several of the isolates showed inhibition of Clostridium perfringens while only a few isolates inhibited E. coli. The strains with the best inhibition of pathogens were primarily of the species B. amyloliquefaciens.

WO2016/060934 shows in Table 7 the anti-E. coli activity of 10 Bacillus strains. Five of the six B. amyloliquefaciens strains demonstrate anti-E. coli activity while only one of the two B. subtilis strains, the strain isolated from Kemin product, clostat, demonstrated anti-E. coli activity. Interestingly, this strain has later been found to be a B. amyloliquefaciens cf. WO2016/118840 (page 46, line 23).

WO2016118840 describes various Bacillus strains for improving health and performance of production animals, in particular two B. amyloliquefaciens strains and two B. subtilis strains (see Table 3.1). Only two of the strains, the B. subtilis strain deposited as DSM29870 and the B. amyloliquefaciens strain deposited as DSM29869, were found to be sensitive for all eight antibiotics tested. Results for ampicillin were not provided.

The B. subtilis strain deposited as DSM29870 was found to inhibit growth of E. coli strains ATCC10535 and ATC25922 in vitro (Example 4). Example 7 provides the results of three Clostridium perfringens challenge trials. There was no significant difference between the DSM29870 fed group (T4) and the bacitracin fed group (T3) with regards to all parameters measured in these trials. Results for BWG, FCR and bird mortality with Necrotic enteritis lesions were intermediate between the non-infected, non-treated group (T1) and the infected, non-treated group (T2).

However, there is still a need for probiotic strains which can be used for improving health and performance of production animals.

SUMMARY OF THE INVENTION

The present invention provides a Bacillus subtilis strain selected from the group consisting of a) the strain deposited as DSM32324, b) the strain deposited as DSM32325, and c) a mutant strain of (a) or (b) which has sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and has inhibitory activity against E. coli and Clostridium perfringens.

A bacterial strain refers to a bacterium which remains genetically unchanged when grown or multiplied. The multiplicity of such identical bacteria is included when reference is made to a strain.

Compositions comprising at least one Bacillus subtilis strain according to the invention, e.g. as Direct Fed Microbial (DFM), an animal feed additive or premix, or an animal feed may be fed to an animal.

At least one Bacillus subtilis strain according to the invention may be added to the feed during production, after production by the supplier or by the person feeding the animal, just prior to providing the feed to the animal. The Bacillus subtilis bacteria used in the methods and compositions described herein are particularly suitable because they are capable of surviving (as spores) the heat and pressure conditions of the process of producing a dry pelleted feed product.

Necrotic enteritis caused by Clostridium perfringens has become a grave economic problem in modern poultry production. The purpose of some of the in vivo studies described in the examples was to investigate the effect of feed additives comprising a Bacillus subtilis strain according to the invention on the pathogenesis of necrotic enteritis (NE) in broiler cage studies. Other in vivo studies in poultry focused on performance with or without challenge with Clostridium perfringens.

Individual battery/floor pen studies have been performed to evaluate the influence of Bacillus subtilis DSM32324 and DSM32325 on the development of subclinical necrotic enteritis. Two different independent research facilities have been used, one based in Europe (Example 3, DSM32324) and the other in the United States (Example 4, DSM32325) meaning that slightly different evaluation parameters were used to assess the effect of the two strains.

For Bacillus subtilis DSM3234 the performance parameters feed conversion rate (FCR) and average weight gain (AWG) measured at day 21, day 35 and day 42 showed a significant improvement for all data points when considering Bacillus subtilis DSM3234 as a feed additive compared to the untreated infected control group. Surprisingly, the Bacillus treated group did not show any significant differences to the not-infected, non-medicated control group, even though the latter group did not receive the challenge and was considered healthy (Table 7).

In the induced subclinical enteritis challenge in vivo trial the Bacillus subtilis DSM3234 decreased necrotic enteritis lesion scoring in chicken and reduced necrotic enteritis mortality significantly (Table 8).

The Bacillus subtilis DSM32325 decreased necrotic enteritis frequency in chicken compared to infected untreated control in a statistically significant manner when the data of Day 25 and 26 were combined. Surprisingly, the necrotic enteritis frequency was even lower in the Bacillus treated group than in the Amoxicilin treated control group (Table 9).

Further, Bacillus subtilis DSM32325 reduced necrotic enteritis severity (mean score) compared to infected untreated control in a statistically significant manner when the data of Day 25 and 26 were combined. Surprisingly, the mean score was even lower in the Bacillus treated group than in the Amoxicilin treated group (Table 10).

The two B. subtilis strains have also been evaluated in two performance feeding trials, Example 5 (DSM32324 and DSM32325) and Example 6 (DSM32324).

Example 5 provides the results of a trial of 1800 Ross 308 male broilers and found that for the global fattening period (0-42 days of age), broilers supplemented with the Bacilli strains DSM32324 or DSM32325 grew significantly more than control animals. The feed conversion (FCR) and EPEF of all broilers supplemented with DSM19489, DSM32324 and DSM32325 were significantly improved when compared to those of the control animals.

Example 6 provides the results of a trial of 1300 Ross 308 male broilers per treatment group and demonstrates that DSM19489 had a tendency of reduced mortality (p=0.096) and Bacillus subtilis DSM32324 a marked and significantly reduced mortality in especially the finisher period which also served for statistical significance of mortality for the overall trial.

Example 7 investigates the effect of three selected Bacilli strains (DSM32324, DSM32325 and DSM25840) on performance and apparent ileal digestibility and concluded that all three strains showed surprisingly good and significantly improved results.

Birds supplemented with DSM32324 showed a higher daily weight gain (Table 16), daily feed intake and feed conversion ratio in the starter period (data not shown) and a higher protein digestibility at D42 (Table 17) compared to non-supplemented birds.

Birds supplemented with DSM25840 showed a higher daily weight gain (Table 16) and daily feed intake in the starter period (data not shown), a higher body weight at D42 (Table 16) and a higher ash, protein and energy digestibility at D42 (Table 17) compared to non-supplemented birds.

Birds supplemented with DSM32325 showed a higher daily weight gain (Table 16) and daily feed intake in the starter period (data not shown), and a lower Ca and Phosphorus digestibility at D42 (Table 17) compared to non-supplemented birds.

In conclusion, these studies have demonstrated that the Bacillus subtilis strains deposited as DSM32324 and DSM32325 show effect on reduction of necrotic enteritis in in vivo challenge trials and positive effect on performance parameters in poultry. Important findings were significant increase of Weight Gain (WG), significant decrease of Feed Conversion Ratio (FCR), significant decrease of mortality and significant increase of European Production Efficacy Factor (EPEF).

Example 8 shows that a Bacillus composition of the invention, EPB5, comprising DSM32324, DSM25840 and DSM32325 in a ratio of 8:3:5, improved performance in broilers when compared to birds fed corresponding diets without addition of the probiotic. Positive responses were demonstrated on feed conversion ratio and average weight gain. Surprisingly, EBP5 treated groups showed significant improvement for some of the performance parameters compared to the groups with the Bacillus single strain treatment which again showed significant differences in performance parameters to the non-medicated infected control group. Further, both the single strains Bacillus subtilis DSM32234, Bacillus subtilis DSM32235 and Bacillus amyloliquefaciens DSM25840 as well as the combination EBP5 decreased necrotic enteritis lesion scoring in chicken and reduced necrotic enteritis mortality significantly in an in vivo challenge trial.

Example 9 shows that a Bacillus composition of the invention improved performance in turkeys from d 1 to d 147 of age (147-d feeding period) at dose levels from 250 mg/kg to 2000 mg/kg when compared to birds fed corresponding diets without addition of the probiotic. Positive responses were demonstrated on body weight gain, feed conversion ratio and dry matter content of excreta.

Example 10 shows that a Bacillus composition of the invention can be combined with a vaccine, such as a live Salmonella Typhimurium vaccine, without affecting the vaccine's initial Salmonella colonization and its subsequent ability to protect against a Salmonella Heidelberg challenge in broiler chicken. The study indicates that there may even be an additive effect to having both the vaccine and the Bacillus composition (Table 20).

Definitions

In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.

As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Animal feed: The term “animal feed” refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal. Animal feed for a monogastric animal comprises concentrates as well as for example vitamins, minerals, enzymes, amino acids and/or other feed ingredients (such as in a premix). The animal feed may further comprise forage. Examples of poultry feed are given in Examples 3 to 7.

Composition: The term “composition” refers to a composition comprising a carrier and at least one bacterial strain as described herein. The compositions described herein may be a Direct Fed Microbial (DFM), an animal feed additive or premix, or an animal feed.

Concentrate: The term “concentrate” means a feed with high protein and energy concentrations, such as fish meal, molasses, oligosaccharides, sorghum, seeds and grains (either whole or prepared by crushing, milling, etc. from, e.g. corn, oats, rye, barley, wheat), oilseed press cake (e.g. from cottonseed, safflower, sunflower, soybean (such as soybean meal), rapeseed/canola, peanut or groundnut), palm kernel cake, yeast derived material and distillers grains (such as wet distillers grains (WDS) and dried distillers grains with solubles (DDGS)).

Control C. perfringens infections and/or necrotic enteritis: The term “control C. perfringens infections and/or necrotic enteritis” means a method and/or composition that partly or completely inhibits C. perfringens infections and/or necrotic enteritis in an animal. Accordingly, the term “control C. perfringens infections and/or necrotic enteritis” means the C. perfringens infections and/or the necrotic enteritis are reduced or completely eliminated.

Direct Fed Microbial: The term “direct fed microbial” or “DFM” means live micro-organisms including spores which, when administered in adequate amounts, confer a benefit, such as improved digestion or health, on the host.

Enzymatic activity under anaerobic conditions: The term “enzymatic activity under anaerobic conditions” means activity of enzymes produced by a Bacillus strain during growth under anaerobic conditions. Example 4 of WO2013/153159 describes a method of 5 testing the Bacillus strains for production of the enzymes xylanase, cellulose and protease.

European Production Efficacy Factor (EPEF): The European Production Efficacy Factor is a way of comparing the live-bird performance of flocks. This single-figure facilitates comparison of performance within and among farms and can be used to assess environmental, climatic and management variables. The EPEF is calculated as [(liveability (%)×Liveweight (kg))/(Age of depletion (days)×FCR)]×100, wherein liveability is the percentage of birds alive at end of study, Liveweight is the average weight gain of the birds at end of study, age of depletion is the age of the birds at end of study and FCR is the feed conversion ratio at end of study.

Effective amount/concentration/dosage: The terms “effective amount”, “effective concentration”, or “effective dosage” are defined as the amount, concentration, or dosage of the bacterial strain(s) sufficient to improve the digestion or yield of an animal. The actual effective dosage in absolute numbers depends on factors including: the state of health of the animal in question, other ingredients present. The “effective amount”, “effective concentration”, or “effective dosage” of the bacterial strains may be determined by routine assays known to those skilled in the art. An example of an effective amount for poultry is given in Examples 3, 4, 5, 6, and 7.

Feed Conversion Rate (FCR): FCR is a measure of an animal's efficiency in converting feed mass into increases of the desired output. Animals raised for meat—such as swine, poultry, cattle, sheep and fish—the output is the mass gained by the animal. Specifically FCR is the mass of the food eaten divided by the output, all over a specified period. FCR can be determined as described in Example 7. Improvement in FCR means reduction of the FCR value.

Feeding an animal: The terms “feeding an animal” or “fed to an animal” means that the composition of the present invention is administered orally to the animal in an effective amount. The oral administration may be repeated, e.g. one or more times daily over a specified time period such as several days, one week, several weeks, one months or several months. Feeding of poultry can, e.g. be performed as described in Examples 3, 4, 5, 6, and 7. Accordingly, the terms “feeding” or “fed” mean any type of oral administration such as administration via an animal feed or via drinking water or, in certain circumstances, by oral gavage or aerosol spray.

Forage: The term “forage” as defined herein also includes roughage. Forage is fresh plant material such as hay and silage from forage plants, grass and other forage plants, seaweed, sprouted grains and legumes, or any combination thereof. Examples of forage plants are Alfalfa (lucerne), birdsfoot trefoil, brassica (e.g. kale, rapeseed (canola), rutabaga (swede), turnip), clover (e.g. alsike clover, red clover, subterranean clover, white clover), grass (e.g. Bermuda grass, brome, false oat grass, fescue, heath grass, meadow grasses, orchard grass, ryegrass, Timothy-grass), corn (maize), millet, barley, oats, rye, sorghum, soybeans and wheat and vegetables such as beets. Forage further includes crop residues from grain production (such as corn stover, straw from wheat, barley, oat, rye and other grains), residues from vegetables like beet tops, residues from oilseed production like stems and leaves from soy beans, rapeseed and other legumes, and fractions from the refining of grains for animal or human consumption or from fuel production or other industries.

Inhibitory activity against Clostridium perfringens: The term “Inhibitory activity against Clostridium perfringens” means that the growth of Clostridium perfringens is inhibited and/or that some or all of the Clostridium perfringens are killed. This can be determined by the assay described in Example 1.

Inhibitory activity against E. coli: The term “Inhibitory activity against E. coli” means that the growth of E. coli is inhibited and/or that some or all of the E. coli are killed. This can be determined by the assay described in Example 1.

Isolated: The term “isolated” means that the bacterial strains described herein are in a form or environment which does not occur in nature, i.e. the strain is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature.

Pellet: The terms “pellet” and/or “pelleting” refer to solid rounded, spherical and/or cylindrical tablets or pellets and the processes for forming such solid shapes, particularly feed pellets and solid extruded animal feed. As used herein, the terms “extrusion” or “extruding” are terms well known in the art and refer to a process of forcing a composition, as described herein, through an orifice under pressure.

Poultry: The term “poultry” means domesticated birds kept by humans for the eggs they produce and/or their meat and/or their feathers. Poultry includes breeders, broilers and layers. Poultry include members of the superorder Galloanserae (fowl), especially the order Galliformes (which includes chicken, Guineafowls, quails and turkeys) and the family Anatidae, in order Anseriformes, commonly known as “waterfowl” and including domestic ducks and domestic geese. Poultry also includes other birds that are killed for their meat, such as pigeons and ostriches. Examples of poultry include chicken (including layers, broilers and chicks), ducks, geese, pigeons, turkeys and quail.

Prevent C. perfringens infections and/or necrotic enteritis: The term “prevent C. perfringens infections and/or necrotic enteritis” means a method and/or composition that prevents development of a C. perfringens infection and/or necrotic enteritis in an animal.

Reducing sugar: A reducing sugar is any sugar that either has a reactive aldehyde group or is capable of forming one to allow the sugar to act as a reducing agent. The reducing ends are formed by the enzymatic cleavage of the glycosidic bond between polymeric carbohydrates. Reducing sugars include glucose, glyceraldehyde and galactose as well as disaccharides, like lactose and maltose and can be measured by the Nelson-Somogyi (NS) or dintrosalicylic acid (DNS) method. DNS is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. The assay simulates the situation when feed is ingested by the animal and is digested in the digestive tract. The ability of different Bacillus strains to degrade Non Starch Polysaccharides (NSP) to reducing sugars has been investigated in Example 2.

Roughage: The term “roughage” means dry plant material with high levels of fiber, such as fiber, bran, husks from seeds and grains and crop residues (such as stover, copra, straw, chaff, sugar beet waste).

Sensitive to antibiotics: The term “sensitive to antibiotics” means the phenotypic property of a bacterial strain, that growth of said bacterial strain is inhibited under conditions where the bacterial strain would otherwise grow. In this context sensitivity to antibiotics is tested after the CLSI guidelines (M07-A8 and M45-A2). A strain of Bacillus is considered sensitive if growth is only detected at or below the breakpoint concentration specified in EFSA Journal 2012; 10(6):2740 for vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol. With regard to ampicillin there is no breakpoint given by EFSA for Bacillus; the breakpoint 4 mg/L has been chosen for a strain to be considered sensitive.

Silage: The term “silage” means fermented, high-moisture stored fodder which can be fed to herbivores such as horses and ruminants e.g. camels, lama, cattle and sheep, or used as a biofuel feedstock for anaerobic digesters. It is fermented and stored in a process called ensilage, ensiling or silaging, and is usually made from grass or cereal crops (e.g. maize, sorghum, oats, rye, timothy, forage grass plants) or legume crops like clovers/trefoils, alfalfa, vetches, using the entire green plant (not just the grain). Silage can be made from many field crops, and special terms may be used depending on type (oatlage for oats, haylage for alfalfa). Silage is made either by placing cut green vegetation in a silo, by piling it in a large heap covered with plastic sheet, or by wrapping large bales in plastic film.

Spore: The terms “spore” and “endospore” are interchangeable and have their normal meaning which is well known and understood by those of skill in the art. As used herein, the term spore refers to a microorganism in its dormant, protected non-reproductive state.

Stable: The term “stable” is a term that is known in the art, and in a preferred aspect, stable is intended to mean the ability of the microorganism to remain in a live form until it is administered to an animal to improve the health of the animal.

Swine: The term “swine” or “pigs” means domesticated pigs kept by humans for food, 20 such as their meat. Swine includes members of the genus Sus, such as Sus scrofa domesticus or Sus domesticus and includes piglets, weaners, growers, finishers, hocks, polts, gilts, sows, and gestation sows.

Vegetable protein: The term “vegetable protein” refers to any compound, preparation or mixture that includes at least one protein derived from or originating from a vegetable, including modified proteins and protein-derivatives.

Vegetable proteins may be derived from vegetable protein sources, such as legumes and cereals, for example materials from plants of the families Fabaceae (Leguminosae), e.g. soybean, lupine, pea, or bean; Cruciferaceae, Chenopodiaceae, e.g. beet, sugar beet, spinach or quinoa; and Poaceae. Other examples of vegetable protein sources are cereals such as barley, wheat, rye, oat, maize (corn), rice, and sorghum.

Weight Gain: The Weight Gain of an animal is the increase of weight of the animal over a specified time period. An example of average Weight Gain determination is given in Example 3 and an example of daily Weight Gain determination is given in Example 7.

DETAILED DISCLOSURE OF THE INVENTION

The present invention provides a Bacillus subtilis strain selected from the group consisting of the strain deposited as DSM32324, the strain deposited as DSM32325, and 5 a mutant strain of DSM32324 or DSM32325 which has sensitivity for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol; and has inhibitory activity against E. coli and Clostridium perfringens.

The bacterial strains described herein are isolated, i.e. present in a form or environment which does not occur in nature.

A “mutant bacterium” or a “mutant strain” refers to a natural (spontaneous, naturally occurring) mutant bacterium or an induced mutant bacterium comprising one or more mutations in its genome (DNA) which are absent in the parent strain DNA. An “induced mutant” is a bacterium where the mutation was induced by human treatment, such as treatment with any conventionally used mutagenization treatment including treatment with chemical mutagens, such as a chemical mutagen selected from (i) a mutagen that associates with or become incorporated into DNA such as a base analogue, e.g. 2-20 aminopurine or an interchelating agent such as ICR-191, (ii) a mutagen that reacts with the DNA including alkylating agents such as nitrosoguanidine or hydroxylamine, or ethane methyl sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV- or gamma radiation etc. In contrast, a “spontaneous mutant” or “naturally occurring mutant” has not been mutagenized by man.

A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5 treatments (or screening/selection steps) are carried out. In a presently preferred mutant less than 1%, less than 0.1, less than 0.01, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced with another nucleotide, or deleted, compared to the mother strain.

Mutant bacteria as described above are non-GMO, i.e. not modified by recombinant DNA technology. As an alternative to the above preferred method of providing the mutant by random mutagenesis, it is also possible to provide such a mutant by site-directed mutagenesis, e.g. by using appropriately designed cloning techniques.

When the mutant is provided as a spontaneously occurring mutant, the strain is subjected to the selection step without any preceding mutagenization treatment.

In one embodiment, the Bacillus subtilis strain of the invention has at least 98% (such as at least 98.5%, such as at least 99%, such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8%, such as at least 99.9%) sequence identity to the nucleotide sequence of DSM32324.

In one embodiment, the Bacillus subtilis strain of the invention has at least 98% (such as at least 98.5%, such as at least 99%, such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8%, such as at least 99.9%) sequence identity to the amino acid sequence of DSM32324.

In one embodiment, the Bacillus subtilis strain of the invention has at least 98% (such as at least 98.5%, such as at least 99%, such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8%, such as at least 99.9%) sequence identity to the nucleotide sequence of DSM32325.

In one embodiment, the Bacillus subtilis strain of the invention has at least 98% (such as at least 98.5%, such as at least 99%, such as at least 99.5%, such as at least 99.6%, such as at least 99.7%, such as at least 99.8%, such as at least 99.9%) sequence identity to the amino acid sequence of DSM32325.

A Bacillus strain is considered to exhibit an inhibitory activity towards E. coli if the inhibition zone is at least 0.5 mm (low inhibition). Preferably, the inhibition zone is at between at least 0.5 mm and 2 mm (medium), more preferably more than 2 mm (high). The inhibition zone may be different for the various E. coli strains. For a strain to be considered to exhibit an inhibitory activity against E. coli according to the present invention it should exhibit an inhibition zone of at least 0.5 mm for all of the E. coli strains tested. Preferably, the inhibition zone of two, three, four or even more preferably the inhibition zone of all five of the E. coli strains is at least between 0.5 mm and 2 mm. Even more preferably, the inhibition zone of two, three, four or even more preferably the inhibition zone of all five of the E. coli strains is more than 2 mm.

A Bacillus strain is considered to exhibit an inhibitory activity towards Clostridium perfringens if the inhibition zone is at least 0.5 mm (low inhibition). Preferably, the inhibition zone is at between at least 0.5 mm and 2 mm (medium), more preferably more than 2 mm (high). The inhibition zone may be different for the various Clostridium perfringens strains. For a strain to be considered to exhibit an inhibitory activity against Clostridium perfringens according to the present invention it should exhibit an inhibition zone of at least 0.5 mm for all of the Clostridium perfringens strains tested. Preferably, the inhibition zone of two, three, four or even more preferably the inhibition zone of all five of the Clostridium perfringens strains is at least between 0.5 mm and 2 mm. Even more preferably, the inhibition zone of two, three, four or even more preferably the inhibition zone of all five of the Clostridium perfringens strains is more than 2 mm.

Preferably, a Bacillus strain should also be able to increase the amount of reducing sugars from degradation of Non Starch Polysaccharides (NSP). The ability of different Bacillus strains to degrade NSP to reducing sugars has been investigated in Example 2 and the results are provided in Table 4. Strains having the ability to increase the available sugar amount to at least 500 kJ/kg feed when tested as outlined in the example are considered preferable.

Based on the detailed assay descriptions the person of ordinary skill in the art is able to repeat these assays to determine whether a specific Bacillus strain complies with the antibiotic sensitivity, the inhibitory activity and the capability of degrading NSP. In this manner the person of ordinary skill in the art will be able to consistently produce strains with the stated properties. Preferably, the person of skill in the art will also include assaying for sensitivity of the vegetative cells at pH 4, and assaying for bile resistance to ensure that the strains are able to survive to a sufficient degree in the gastrointestinal tract e.g. as described in WO2013/153159. Evidently, these assays can be performed in any order and some strains may be excluded during the process if they do not fulfill the criteria.

The invention further provides a Bacillus composition comprising at least one Bacillus subtilis strain of the invention. In one embodiment, the Bacillus composition comprises one Bacillus subtilis strain of the invention. In another embodiment, the Bacillus composition comprises two Bacillus subtilis strains of the invention, for example a combination of Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

The Bacillus compositions according to the invention may comprise a combination of at least one of the Bacillus subtilis strains of the invention and at least one other Bacillus strain. The Bacillus composition may comprise at least two strains, such as at least three, such as at least four, such as at least five Bacillus strains, at least one of which is a Bacillus subtilis strain of the present invention.

Bacillus strains may be used in any combination in the Bacillus compositions. For example, the Bacillus composition may comprise at least one Bacillus subtilis strain of the invention and/or at least one Bacillus licheniformis strain and/or at least one Bacillus amyloliquiefaciens strain e.g. two Bacillus subtilis strains of the invention and at least one Bacillus amyloliquiefaciens strain. The composition may comprise Bacillus subtilis DSM32324 and/or Bacillus subtilis DSM32325 in combination with Bacillus amyloliquiefaciens DSM25840 and/or Bacillus licheniformis DSM17236 and/or Bacillus subtilis DSM19489. Any other possible combination of the Bacillus strains of the present invention with other Bacillus strains may also be made. As a specific example, the Bacillus composition comprises Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus amyloliquiefaciens DSM25840. As another specific example, the Bacillus composition comprises Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus licheniformis DSM17236. In a yet further specific example, the Bacillus composition comprises Bacillus subtilis DSM32324, Bacillus subtilis DSM32325 and Bacillus subtilis DSM19489.

If more than one strain is used, it is contemplated that the proportion of each strain in the composition will be 1 to 99%, such as 20 to 80%, e.g. 30 to 70%, more particularly 20%, 33%, 40% or 50% of the total amount of bacterial isolates calculated as CFU/g composition. The individual strains may be present in about equal numbers or in unequal numbers.

A presently preferred embodiment of the invention is a Bacillus composition comprising DSM32324, DSM25840 and DSM32325 in a ratio of 8:3:5.

The relevant Bacillus strain or strains are provided in a commercially relevant form known to the skilled person. Accordingly, in an embodiment the Bacillus strain or strains of the composition are present in a dried (e.g. spray dried) or frozen form. The composition may be provided in any suitable form such as in the form of a liquid e.g. a gel, a slurry, a powder or a pellet.

In a preferred embodiment, the Bacillus composition comprises from 10⁵ to 10¹² CFU/g, such as from 5×10⁵ to 10¹² CFU/g, more preferably from 10⁶ to 10¹² CFU/g, and most preferably from 10² to 10¹² CFU/g, such as from 10⁸ to 10¹¹ CFU/g, e.g. from 10⁹ to 10¹⁰ CFU/g of each of the bacterial strains in the composition. The Bacillus composition comprises at least 5×10⁴ CFU of each strain per gram of the composition which distinguishes a composition of the present invention from e.g. animal feed with naturally occurring strains.

The term “CFU/g” relates to the gram weight of the composition including carriers such as calcium carbonate, anti caking agents such as aluminum silicates and kieselgur (diatomaceous earth), and other components present in the composition.

Compositions of the present invention include at least one Bacillus strain of the invention and at least one carrier and/or other component that make the composition suitable for feeding to an animal or as an additive for drinking water.

As used herein the term “premix” refers to a Bacillus strain added to a carrier to make a premix which is then added to an animal feed at a desired inclusion rate.

Alternatively, at least one Bacillus strain of the invention may be formulated with animal feed ingredients as discussed in detail in the following. Such combinations may be in the form of pellets that are extruded through standard pelleting processes.

The invention also provides a method for producing an animal feed, animal feed additive or premix comprising adding at least one Bacillus strain of the invention to an animal feed or relevant components thereof.

Bacillus bacteria exist as spores and vegetative cells which can divide to produce more vegetative cells. When reference is made herein to Bacillus, this relates to both spores and vegetative cells unless the context indicates otherwise.

In the Bacillus composition of the present invention, the Bacillus strain or strains are preferably provided as spores. The primary function of sporulation is generally to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, starvation, and chemical disinfectants. The spore coat is impermeable to many toxic molecules and may also contain enzymes that are involved in germination. The core has normal cell structures, such as DNA and ribosomes, but the spore is metabolically inactive.

The vegetative form of the bacteria produces effectors which may reduce bacterial pathogens or have other beneficial effects in the gastrointestinal tract of an animal. Thus, reactivation and germination of the spores after administration to the animal is important.

It is known from the literature that bile has some negative influences on the survival and germination and outgrowth to vegetative cells in the GIT of animals. Therefore probiotic bacteria shall generally be able to survive and proliferate in the gut of animals by being able to tolerate a low pH and resistant to bile salt in order to be useful as probiotic Bacillus compositions for the addition to animal feed. The examples provide useful in vitro tests in this regard. The test for sensitivity to low pH (simulating gastric conditions) focuses on the resistance of vegetative cells to pH 4. It is well known that spores are resistant at pH values of 2-3 and that vegetative cells will die at pH 2. However, gastric pH may have pH values of up to 4 especially in feeding conditions. This may result in germination of the spores and it is thus relevant to test the sensitivity of vegetative cells at pH 4. Selected strains should preferably be resistant to pH at 4.

The Bacillus subtilis strains deposited as DSM32324 and DSM32325 have been assayed for sensitivity of the vegetative cells at pH 4 and bile resistance to ensure that the strains are suitable.

A Bacillus composition may comprise one or two different strains of B. subtilis and/or one or two strains of B. licheniformis and/or one or two strains of Bacillus amyloliquiefaciens, wherein each strain is independently selected to perform a specific role and/or function. The combination of these strains can be combined with an animal feed, such as poultry feed, and ultimately used to improve the health and productivity of the agricultural animals (e.g. livestock and/or poultry). For example the strains and/or the combined strains can reduce gut pathogens in the poultry and increase weight gain of commercial poultry.

In a further embodiment, the Bacillus composition of the invention can be combined with a vaccine, such as a live Salmonella Typhimurium vaccine, to reduce infection by Salmonella and/or increase FCR.

In one aspect, the present invention provides an an animal feed, animal feed additive or premix comprising at least one Bacillus subtilis strain according to the invention, and further comprising one or more of concentrate(s), vitamin(s), mineral(s), enzyme(s), amino acid(s) and/or other feed ingredient(s). In one embodiment the animal feed, animal feed additive or premix comprises the Bacillus subtilis strain DSM32324. In another embodiment the animal feed, animal feed additive or premix comprises the Bacillus subtilis strain DSM32325. The animal feed, animal feed additive or premix may comprise both Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

In a specific embodiment, the animal feed comprises forage. Generally, the forage comprises a vegetable protein source. In a particular embodiment, the vegetable protein source is material from one or more plants of the family Fabaceae. In another particular embodiment, the vegetable protein source is material from one or more plants of the family Chenopodiaceae. Other examples of vegetable protein sources are rapeseed and cabbage. In another particular embodiment, soybean is a preferred vegetable protein source. As an example, the forage comprises 0-80% maize; and/or 0-80% sorghum; and/or 0-70% wheat; and/or 0-70% barley; and/or 0-30% oats; and/or 0-40% soybean meal; and/or 0-10% fish meal; and/or 0-20% whey.

In one embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with a concentrate. In another embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with a premix. In a further embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with vitamins and/or minerals. In a further embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with one or more enzymes. In a further embodiment, the forage and at least one Bacillus subtilis strain of the invention are mixed with other feed ingredients, such as colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides, anti-fungal polypeptides and amino acids.

In a particular embodiment, the animal feed consists of or comprises milk (e.g. from sow, cow, goat, sheep), e.g. for feeding of piglets. In another particular embodiment, the animal feed consists of or comprises milk replacement, e.g. for feeding of piglet.

In another embodiment, the animal feed may include one or more vitamins, such as one or more fat-soluble vitamins and/or one or more water-soluble vitamins. In another embodiment, the animal feed may optionally include one or more minerals, such as one or more trace minerals and/or one or more macro minerals. Usually fat- and water-soluble vitamins, as well as trace minerals form part of a so-called premix intended for addition to the feed, whereas macro minerals are usually separately added to the feed. Non-limiting examples of fat-soluble vitamins include vitamin A, vitamin D3, vitamin E, and vitamin K, e.g. vitamin K3. Non-limiting examples of water-soluble vitamins include vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate. Non-limiting examples of trace minerals include boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium and zinc. Non-limiting examples of macro minerals include calcium, magnesium, potassium and sodium.

The animal feed, animal feed additive or premix of the invention may also comprise at least one enzyme selected from the group comprising of phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 5 3.2.1.22); protease (EC 3.4); phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC 3.2.1.17); and beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6), or any mixture thereof.

The animal feed, animal feed additive or premix of the invention may further comprise one or more added amino acids. Examples of amino acids which are used in animal feed are lysine, alanine, beta-alanine, threonine, methionine and tryptophan. The animal feed, animal feed additive or premix of the invention may further comprise colouring agents, stabilisers, growth improving additives and aroma compounds/flavorings, polyunsaturated fatty acids (PUFAs); reactive oxygen generating species, anti-microbial peptides and anti-fungal polypeptides. Examples of colouring agents are carotenoids such as beta-carotene, astaxanthin, and lutein. Examples of aroma compounds/flavorings are creosol, anethol, deca-, undeca-and/or dodeca-lactones, ionones, irone, gingerol, piperidine, propylidene phatalide, butylidene phatalide, capsaicin and tannin. Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid. Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

In one embodiment the animal feed, animal feed additive or premix comprises one or more coccidiostats.

The animal feed, animal feed additive or premix further comprises a carrier. The carrier can comprise one or more of the following compounds: water, glycerol, ethylene glycol, 1,2-propylene glycol or 1,3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, whey, whey permeate, wheat flour, wheat bran, corn gluten meal, starch and cellulose.

In one embodiment, the one or more bacterial strains are stable when subjected to pressures applied/achieved during an extrusion process for pelleting. In a particular embodiment, the one or more bacterial strains are stable at pressures ranging from 1 bar to 40 bar.

In a particular embodiment, the one or more bacterial strains are stable at high temperatures. In particular, the bacterial strains are stable when they are subjected to temperatures achieved during an extrusion process for pelleting. In an even more particular embodiment, the one or more bacterial strains are stable at temperatures ranging from 70° C. to 120° C.

In an embodiment, the animal feed, animal feed additive or premix further comprises one or more additional microorganisms. In a particular embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus, Pediococcus, Enterococcus, Leuconostoc, Carnobacterium, Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or any combination thereof.

In a particular embodiment, the animal feed, animal feed additive or premix further comprises a bacterium from one or more of the following strains of Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus pumilus, Bacillus polymyxa, Bacillus licheniformis, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus simplex, Bacillus mojavensis, Bacillus safensis, Bacillus simplex, Bacillus atrophaeus, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis, Bacillus tequilensis. or any combination thereof.

In a particular embodiment, the animal feed, animal feed additive or premix further comprises one or more types of yeast. The one or more types of yeast can be selected from the group consisting of Saccharomycetaceae, Saccharomyces (such as S. cerevisiae and/or S. boulardii), Kluyveromyces (such as K. marxianus and K. lactis), Candida (such as C. utilis, also called Torula yeast), Pichia (such as P. pastoris), Torulaspora (such as T. delbrueckii), Phaffia yeasts and Basidiomycota.

Animal diets can, e.g. be manufactured as mash feed (non-pelleted) or pelleted feed. Typically, the milled feed-stuffs are mixed and sufficient amounts of essential vitamins and minerals are added according to the specifications for the species in question. The bacteria cultures and optionally enzymes can be added as solid or liquid formulations. For example, for mash feed a solid or liquid culture formulation may be added before or during the ingredient mixing step. For pelleted feed the (liquid or solid) Bacillus composition may also be added before or during the feed ingredient step. Typically a liquid Bacillus composition of the invention comprises the bacterial strain(s) optionally with a polyol, such as glycerol, ethylene glycol or propylene glycol, and is added after the pelleting step, such as by spraying the liquid formulation onto the pellets. The bacteria may also be incorporated in an animal feed additive or premix.

The composition according to the invention may be used for the prevention or control of a bacterial colonization or infection, e.g. by E. coli and/or Clostridium, such as Clostridium difficile, Clostridium novyi, Clostridium perfringens, or Clostridium septicum.

In another aspect, the invention relates to a method for the prevention or control of a bacterial colonization or infection, e.g. by E. coli and/or Clostridium, such as Clostridium difficile, Clostridium novyi, Clostridium perfringens, or Clostridium septicum, the method comprising administering an effective amount of a strain according to the invention or a composition according to the invention to an animal in need thereof.

Another aspect of the invention relates to a method for feeding an animal comprising administering a Bacillus composition of the invention to an animal, in particular a monogastric animal.

Monogastric animals include, but are not limited to, poultry such as broilers, breeders, layers, turkey, ostriches, quails, ducks, and geese, herbivores, such as horses and ruminants, e.g. camels, lamas, cattle and sheep, calves, swine, such as piglets, weaners, growers, finishers, hocks, polts, gilts, sows, gestation sows, rodents such as rabbits, pets such as cats and dogs and fish (including but not limited to salmon, trout, tilapia, catfish and carps; and crustaceans (including but not limited to shrimps and prawns). Pigs and/or poultry are preferred monogastric animals.

In another aspect, the invention relates to the use of at least one Bacillus subtilis strain of the invention or an animal feed, animal feed additive or premix comprising at least one Bacillus subtilis strain of the invention to improve the performance of an animal, in particular a monogastric animal

As evidenced in the examples, administration of a Bacillus subtilis strain of the invention improves the gastrointestinal health of the animal, e.g. prevent or control enteritis, and provides improved animal performance parameters for the treated animals as compared to controls. Animal performance parameters include but are not limited to weight gain (WG), feed conversion rates (FCR), decrease of mortality and increase of European Production Efficacy Factor (EPEF).

The invention further provides a method of increasing digestibility of an animal feed, such as protein digestibility, the method comprising feeding a strain according to the invention or a composition according to the invention to an animal.

Accordingly, the invention relates to use of a strain according to the invention, or a composition according to the invention, for improving one or more animal performance parameters selected from the group consisting of:

-   -   i) increased weight gain (WG),     -   ii) lower feed conversion ratio (FCR),     -   iii) lower necrotic enteritis lesion scoring,     -   iv) lower necrotic enteritis frequency,     -   v) lower necrotic enteritis mortality,     -   vi) increased European Production Efficacy Factor (EPEF), and     -   vii) lower mortality.

In a preferred embodiment of the invention, “animal performance” is determined by the body weight gain of the animal and/or by the feed conversion ratio. By “improved animal performance” it is meant that there is increased body weight gain and/or reduced feed conversion ratio and/or improved digestibility of nutrients or digestible energy in a feed and/or metabolizable energy and/or increased feed efficiency resulting from the use of animal feed, animal feed additive or premix of the present invention in animal feed in comparison to animal feed which does not comprise said animal feed, animal feed additive or premix. Preferably, by “improved animal performance” it is meant that there is increased body weight gain and/or reduced feed conversion ratio.

An “increased weight gain” refers to an animal having increased body weight on being fed feed comprising a feed composition compared with an animal being fed a feed without said feed composition of the invention. Specifically, the Weight Gain (WG) of an 30 animal is the increase of weight of the animal over a specified time period. In one embodiment, the improvement in body weight gain is of at least 0.5%, such as at least 1%, such as at least 2%, such as at least 2.5%, such as at least 3%, such as at least 4%, such as at least 5%, such as at least 6%, such as at least 7%, such as at least 8%, such as at least 9%, such as at least 10%.

In one embodiment, the improvement in weight gain results in a body weight gain of at least 0.5%, such as at least 0.8%, such as at least 1.2%, such as at least 1.5%, such as at least 1.8%, such as at least 2.0%, such as at least 2.5%, such as at least 3.0%, such as at least 4.0%, such as at least 5.0%, such as at least 6.0%, such as at least 7.0%. In a preferred embodiment, the improvement in weight gain results in a weight gain selected from the group consisting of from 1.8% to 2.0%, from 2.0% to 2.2%, from 2.2% to 2.4%, from 2.4% to 2.6%, from 2.6% to 2.8%, from 2.8% to 3.0%, from 3.0% to 3.2%, from 3.2% to 3.4%, from 3.4% to 3.6%, from 3.6% to 3.8%, from 3.8% to 4.0%, from 4% to 5%, from 5% to 7%, from 7% to 10%, or any combination thereof.

By “lower feed conversion ratio” or “improved feed conversion ratio” it is meant that the use of a feed additive composition in feed results in a lower amount of feed being required to be fed to an animal to increase the weight of the animal by a specified amount compared to the amount of feed required to increase the weight of the animal by the same amount when the feed does not comprise said feed additive composition.

In one embodiment, the improvement of feed conversion ratio (FCR) results in a FCR of −2.5% or less than −2.5%, such as less than −2.6%, such as less than −2.7%, such as less than −2.8%, such as less than −2.9%, such as less than −3.0%. In a preferred embodiment, the improvement of FCR results in a FCR of from −5% to −2%, such as a FCR of from −4% to −2%, such as a FCR of from −3.5% to −2.5%. In a specific embodiment, the improvement of FCR results in a FCR within an interval selected from the group consisting of from −5% to −4.5%, from −4.5% to −4%, from −4% to −3.8%, from −3.8% to −3.6%, from −3.6% to −3.4%, from −3.4% to −3.2%, from −3.2 to −3.0%, from −3.0% to −2.8% and from −2.8 to −2.5%, or any combination of these intervals.

Nutrient digestibility as used herein means the fraction of a nutrient that disappears from the gastrointestinal tract or a specified segment of the gastrointestinal tract, e.g. the small intestine. Nutrient digestibility may be measured as the difference between what is administered to the subject and what comes out in the faeces of the subject, or between what is administered to the subject and what remains in the digesta on a specified segment of the gastrointestinal tract, e.g. the ileum. Nutrient digestibility as used herein may be measured by the difference between the intake of a nutrient and the excreted nutrient by means of the total collection of excreta during a period of time; or with the use of an inert marker that is not absorbed by the animal, and allows the researcher calculating the amount of nutrient that disappeared in the entire gastrointestinal tract or a segment of the gastrointestinal tract. Such an inert marker may be titanium dioxide, chromic oxide or acid insoluble ash. Digestibility may be expressed as a percentage of the nutrient in the feed, or as mass units of digestible nutrient per mass units of nutrient in the feed. Nutrient digestibility as used herein encompasses starch digestibility, fat digestibility, protein digestibility, mineral digestibility and amino acid digestibility.

In another embodiment, the invention relates to a method of improving one or more animal performance parameters selected from the group consisting of

-   -   i) increased weight gain (WG),     -   ii) lower feed conversion ratio (FCR),     -   iii) lower necrotic enteritis lesion scoring,     -   iv) lower necrotic enteritis frequency,     -   v) lower necrotic enteritis mortality,     -   vi) increased European Production Efficacy Factor (EPEF), and     -   vii) lower mortality,

the method comprising feeding a strain according to the invention or a composition according to the invention to an animal.

A composition of the present invention may also be used for flexible feed formulation (FFF) wherein an animal is being fed with a feed having a reduced metabolizable energy and a composition of the invention whereby an acceptable animal performance and or feed conversion ratio is obtained in spite of the reduced metabolizable energy in the feed. The reduced metabolizable energy may be in the level of from 97% to 99% of standard feed for the animal in question, such as from 97% to 98% or from 98% to 99%.

Deposit and Expert Solution

The Bacillus licheniformis strain DSM17236 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Apr. 7, 2005 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis strain DSM19489 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Jun. 27, 2007 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus mojavensis strain DSM25839 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus amyloliquefaciens strain DSM25840 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis strain DSM25841 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Apr. 3, 2012 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus amyloliquefaciens strain DSM27032 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Mar. 21, 2013 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis strain DSM32324 has been deposited at DSMZ (Deutsche Sannnnlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Jun. 8, 2016 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

The Bacillus subtilis strain DSM32325 has been deposited at DSMZ (Deutsche Sannnnlung von Mikroorganismen und Zellkulturen GmbH, Inhoffenstrasse 7B, D-38124 Braunschweig) on Jun. 8, 2016 by Chr. Hansen A/S, Denmark. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

For all of the above-identified deposited microorganisms, the following additional indications apply:

As regards the respective Patent Offices of the respective designated states, the applicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn.

IN VITRO EXAMPLES Example 1 Screening for Pathogen Inhibition and Antibiotic Sensitivity Materials

Veal Infusion Broth (VIB) (Difco, 234420)

Veal Infusion Broth (VIB) agar (VIB +1.5% Agar bacteriological (Agar no. 1), Oxoid LP0011)

Muller Hinton Broth 2, Cation-adjusted (Fluka)

T3 agar plates (per liter: 3 g of tryptone, 2 g of tryptose, 1.5 g of yeast extract, 0.05 M sodium dihydrogen phosphate and 0.005 g of MnCl2 [pH 6.8], and 15 g agar)

Laura-Bertani (LB) broth (g/L: Bacto tryptone 10 (Difco 0123), Yeast extract 5 (Oxoid 35 L21), NaCl 10 (Merck nr. 106404))

Brain Heart Infusion (BHI) agar (Oxoid CM375)

Bile salts (Bile extract, porcine; Sigma B8631)

Bioassay dishes (Nunc 240845)

Petri dishes (Procudan 140096, petridish with ribs)

Physiological saline solution with peptone (0.9% sodium chloride, 1% peptone) FKP ISO-SENSITEST Broth (Oxoid CM0473)

Microtitre plates (MTP) NUNC, Denmark

Omni tray/single well plates N 242811 Thermo Scientific/NUNC Denmark

Deep well microtitre 96 well trays (DW) Rnase/DNase free (Thermo Fisher Science)

Ampicillin (Sigma, A9518-5G)

Vancomycin (Sigma, V1764-250MG)

Gentamicin (Sigma, G1264-50MG)

Kanamycin (Sigma, K1377-1G)

Streptomycin (Sigma, 56501-5G)

Erythromycin (Sigma E-5389)

Clindamycin (Sigma, C2569-10MG)

Tetracycline (Sigma T-7660)

Chloramphenicol (Sigma, C0378-5G)

Escherichia coli 0101 H-, K99 F5 (State Serum Institute, Copenhagen, Denmark)

Escherichia coli 0147:K89 F4 H19 (State Serum Institute, Copenhagen, Denmark) 20 Escherichia coli 0149:k91,k88a,c,h10 NCTC10650, (National Collection of Type Cultures, England)

E. coli ATCC11775 (American type culture collection)

E. coli Cp6salp3 (Copenhagen Veterinary University)

Clostridium perfringens Type A, DSM756, Leibniz-Institut DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen

Clostridium perfringens type C, NCTC3180, National Collection of Type Cultures (England)

Clostridium perfringens CCUG2036 (Culture Collection, University of Gothenburg, Sweden)

Clostridium perfringens CCUG2037 (Culture Collection, University of Gothenburg, Sweden)

Clostridium perfringens CCUG44727 (Culture Collection, University of Gothenburg, Sweden)

All pathogen strains mentioned above were maintained in LB with 20% glycerol in BHI at 35 -80° C.

Bacillus Cultures

Bacillus strains isolated from feces, soil, food sources and collected from strain bank collections were maintained in VIB with 20% glycerol in MTP master plates at −80° C.

Bacteria spore-forming aerobic isolates were subjected to identification by 16S ribosomal sequence and gyrB (Wang et al., 2007), screening for antibiotic susceptibility according to “Guidance on the assessment of bacterial susceptibility to inhibitorys of human and veterinary importance.” EFSA Journal 2012;10(6):2740 as described below, and bile resistance and sensitivity to low pH, enzymatic activity, growth in different media, heat resistance and sporulation as described in WO2013/153159.

Antibiotic Susceptibility Measured by MIC

Bacillus strains were analyzed for antibiotic susceptibility by measuring the minimum 10 inhibitory concentration (MIC) for a number of antibiotics. The method used was a broth microdilution method as outlined by the standard of CLSI (Clinical and Laboratory Standards Institute M07-A8 and M45-A2).

A suspension of an overnight culture of the strain to be tested was inoculated in ISO-SENSITEST Broth (Oxoid CM0473) in microtitre plates at an approximate concentration of 10⁵ CFU/ml (colony-forming units/ml) in two-fold serial dilutions of the antibiotic to be tested (total volume 100 μl/well) and incubated aerobically for 20-24 hours at 37° C. The results were recorded after 20 hours of incubation as the lowest concentration of the antibiotic to inhibit visible growth. The test was performed twice as two independent biological replicates.

Only Bacillus strains which were susceptible to inhibitorys according to the EFSA Guidance were included in the screening for inhibition of pathogenic E. coli and Clostridium perfringens.

Screening of Bacillus Strains for Inhibition of Pathogenic E. coli

Bacillus strains were added in a volume of 50 pl from MTP master plates into 700 μl VIB in DW plates and incubated at 37° C. and 175 rpm overnight. E. coli strains were grown in LB at 30° C. overnight. 2 ml of E. coli overnight culture was mixed with 200 ml liquid VIB agar at 50° C., and poured into each bioassay dish. The dishes were dried in a sterile bench. Overnight Bacillus cultures, 2 pl of each, were spotted onto the surface of the VIB agar mixed with E. coli in the bioassay dishes and incubated at 37° C. for 1 day.

Radii of clarified inhibition zones around the Bacillus were measured and recorded as “3 =high”—more than 2 mm, “2=medium”—between 0.5-2 mm and “1=low”—less than 0.5 mm and 0=no inhibition.

Clostridium Perfringens Inhibition by Agar Spot Test

VIB agar was poured into bioassay dishes (200 ml per dish) and dried thoroughly in a sterile bench. Overnight Bacillus cultures, 2 μl of each, were spotted onto the surface of the VIB agar dishes and incubated at 37° C. overnight. Clostridium perfringens strains were grown anaerobically on BHI agar at 37° C. overnight. Overnight culture of Clostridium perfringens was added in a volume of 2 ml to 200 ml liquid BHI agar, mixed and overlaid gently into the bioassay dishes with Bacillus spots. The dishes were incubated anaerobically at 37° C. for 1 day.

Radii of clarified inhibition zones around the Bacillus were measured and recorded as “3 =high”—more than 2 mm, “2=medium”—between 0.5-2 mm and “1=low”—less than 0.5 mm and 0=no inhibition.

All data were replicated on separate days.

Results

TABLE 1 Results of selected Bacillus strains' inhibition of E. coli E. coli inhibition E. coli Inhibition E. coli E. coli E. coli ATCC E. coli DSM number if available O101 F5 O147:K89 F4 O149:k91, k88a 11775 Cp6salp3 Bacillus 0 0 0 0 0 licheniformis Bacillus 17236 0 0 0 0 0 licheniformis Bacillus subtilis 19489 0 0 0 0 0 Bacillus subtilis 0 0 1 1 1 Bacillus subtilis 32324 3 3 3 3 3 Bacillus subtilis 0 0 0 0 0 Bacillus subtilis 0 0 0 0 0 Bacillus subtilis 32325 2 2 3 2 2 Bacillus subtilis 25841 2 2 2 2 2 Bacillus 25840 1 1 2 1 1 amyloliquefaciens Bacillus 27032 2 3 3 2 2 amyloliquefaciens

TABLE 2 Results of selected Bacillus strains' inhibition of Clostridium Inhibition Inhibition DSM number if available DSM756 NCTC3180 CCUG2036 CCUG2037 CCUG44727 Bacillus 1 2 1 0 1 licheniformis Bacillus 17236 1 1 0 0 0 licheniformis Bacillus subtilis 19489 0 0 0 1 0 Bacillus subtilis 0 2 0 0 1 Bacillus subtilis 32324 3 3 3 3 3 Bacillus subtilis 1 0 1 2 1 Bacillus subtilis 1 0 0 1 0 Bacillus subtilis 32325 3 3 3 2 3 Bacillus subtilis 25841 2 3 2 2 3 Bacillus 25840 2 3 3 2 3 amyloliquefaciens Bacillus 27032 3 3 3 2 3 amyloliquefaciens

The results in Tables 1 and 2 of the inhibition of E. coli and Clostridium show that the Bacillus licheniformis strains and many of the Bacillus subtilis strains tested did not demonstrate any E. coli inhibition and poor Clostridium inhibition. However, the two B. subtilis strains DSM32324 and DSM32325 demonstrated impressive results with regard to both E. coli inhibition and Clostridium inhibition.

Example 2 Measurement of Amount of Reducing Sugars in Feed Incubated with a Bacillus Composition

The objective of this experiment was to examine the ability of different Bacillus strains to degrade NSP in commercial poultry starter feed and increase the available sugar amount.

TABLE 3 Composition of compound feed used in the assay Ingredient % of feed ration Ground Corn 30.0 Wheat 27.0 Soy Bean Meal 22.5 Rape seed 6.0 Sunflower 5.0 Oat 4.0 Fish meal 2.0 Limestone 1.24 Monocalciumfosfat 0.78 Vegetable oil 0.54 Sodium bicarbonate 0.28 Vitamin, mineral, amino acid pre- 0.25 MIX Sodium chloride (0.17%); 0.17

Compound feed based on wheat-corn-soybean (Table 3) was autoclaved at 121° C. for 15 min for sterilization. Then the feed sample was diluted 20 fold with sodium phosphate buffer to ensure a pH at about 6-6.5 throughout the whole experiment. Bacillus products were obtained by inoculation with 2% overnight culture of the Bacillus strains, grown in Veal Infusion Broth (VIB) (Difco, 234420). A sample was taken for analysis for reducing sugar (DNS) (T=0). After incubation at 37° C. for 24 hours, a sample was taken for CFU determination. Another sample was centrifuged and the supernatant used for determining DNS.

Reducing sugar was analyzed by 3.5-dinitrosalicylic acid (DNS) assay as follows: Na-acetate buffer (100 mM, pH 6) was mixed with sterile filtered Bacillus sample supernatant and incubated at 40° C. for 10 min. DNS reagent was added to the test tube, mixed and incubated in a boiling water bath for 5 min. After cooling, absorbance was measured at 540 nm in a spectrophotometer.

A standard curve was established with a glucose stock solution for presenting results in reducing sugar or enzyme units (amount of enzyme needed to release 1 μmol reducing glucose equivalent in 1 ml per time unit).

The results are shown in Table 4.

Results

TABLE 4 Sample DSM number if kJ/kg available feed Control 214 Bacillus aryabhattai 237 Bacillus licheniformis 17236 340 Bacillus licheniformis 15326 541 Bacillus subtilis 19489 403 Bacillus subtilis 25841 458 Bacillus subtilis 32325 642 Bacillus subtilis 32324 723 Bacillus 16734 515 amyloliquefaciens Bacillus 27032 515 amyloliquefaciens Bacillus 14623 517 amyloliquefaciens Bacillus 15509 853 amyloliquefaciens Bacillus 25840 1142 amyloliquefaciens Bacillus mojavensis 25839 939

Table 4 shows the results of a number of different Bacillus strains and shows that all Bacillus strains tested supplied more nutrients to the animal by delivering more reducing sugars compared to control but also that there is considerable variance between the individual strains.

Based upon the results of E. coli and Clostridium perfringens inhibition combined with the results of the ability to provide an increased amount of reducing sugars the two best performing strains, the B. subtilis strains DSM32324 and DSM32325, were selected for in vivo studies.

IN VIVO EXAMPLES Example 3 Efficiacy of the Bacillus Subtilis Strain DSM32324 in Clostridium Perfringens Challenge in VIVO TRIALS

TABLE 5 Diets Ingredient Name % (w/w) starter grower finisher Corn, grain 58.509 64.054 69.218 Soybean meal, dehulled, solvent 35.550 29.771 24.511 Fat, vegetable 2.100 2.585 2.748 Dicalcium phosphate 1.734 1.780 1.693 Calcium carbonate 1.150 0.910 0.873 Salt, (NaCl) 0.386 0.390 0.393 Methionine MHA, L—Lysine, Trace Minerals, Vitamin premix and L-Threonine were included accordingly to the breeder's recommendations.

A non-medicated corn/soya bean meal based diet (Table 5) was used. Bacillus subtilis DSM32324 1.2 10⁶ CFU/g was added to the feed of one of the groups. Feed and water were ad libitum available throughout all trials. All feed was by pen. Starter feed was fed from day 0 to 21. On day 21, non-consumed starter diet was weighed and discarded. Grower feed was fed until day 35 and non-consumed grower feed was weighed and discarded. Likewise, finisher feed was fed until day 42 where non-consumed finisher diet was weighed and discarded.

TABLE 6 Clostridium perfringens CP-6 Treatment Inoculation Pens/Trt birds/Trt Non-medicated Not infected 8 320 Non-medicated Day 19, 20 and 21 8 320 DSM32324 Day 19, 20 and 21 8 320

The experiment started with 40 male Ross 308 broiler chickens per pen. The treatments were replicated in eight blocks, randomized within blocks of six pens each.

On Days 19, 20, and 21, all pens, except the non-medicated not infected treatment 1 group, were challenged with a broth culture of C. perfringens CP-6 (Knap I, et al., 2010).

This strain is a field isolate of C. perfringens known to cause NE originating from a commercial broiler operation and utilized in the present study as the challenge organism. Fresh inoculum was used each day. Each pen received the same amount of inoculum corresponding to approximately 1×10⁸ to 1×10⁹ CFU of C. perfringens CP-6. The inoculum was administered by mixing into the feed in the base of the tube feeders.

On Day 21, three birds from each pen were selected, sacrificed, group weighed, and examined for the degree of presence of necrotic enteritis lesions. The scoring was based on a 0 to 3 score, with 0 being normal and 3 being the most severe. The scoring was as follows: 0 for normal intestines, 1 for slight mucus covering and loss of tone, 2 for severe necrotizing enteritis, and 3 for extreme necrotizing enteritis with presence of blood in the lumen.

All birds were weighted at day 31, 35 and 42 to see the impact of necrotic enteritis on performance parameters: Means for live weight, average weight gain (AWG), feed consumption, feed conversion ratio (FCR), Necrotic enteritis lesion scores, and mortality (total and NE) were calculated for all pens.

Statistical data analysis was performed in SAS Stat Version 9.2 using ANOVA analysis with complete randomized design to establish differences between treatment groups. Pen was considered the statistical experimental unit with diets as fixed effect. Results were reported as least square means and were assumed different at P<0.05.

Results

TABLE 7 Treatment FCR AWG (kg) Day 21 Non-medicated, not infected 1.447c 0.562a Non-medicated, infected 1.652a 0.501b DSM32324, infected 1.575b 0.567a Day 35 Non-medicated, not infected 1.569b 1.735ab Non-medicated, infected 1.634a 1.679b DSM32324, infected 1.593b 1.756a Day 42 Non-medicated, not infected 1.627b 2.272a Non-medicated, infected 1.731a 2.155b DSM32324, infected 1.645b 2.303a

The letters adherent to the results represent treatment groups statistically significantly different from non-medicated control (P 0.05) or from each other.

TABLE 8 NE NE % Treatment Lesions scoring Mortality Non-medicated, not infected 0.05b 0.0b Non-medicated, infected 0.58a 4.3a DSM32324, infected 0.18b 0.7b

The letters adherent to the results represent treatment groups statistically significantly different from non-medicated control (P 0.05) or from each other.

Conclusion

For the performance parameters feed conversion rate (FCR) and average weight gain

(AWG) measured at day 21, day 35 and day 42, a significant improvement was seen for all data points when considering Bacillus subtilis DSM3234 as a feed additive compared to the untreated infected control group. Surprisingly, the Bacillus treated group did not show any significant differences to the not-infected, non-medicated control group, even though the latter group did not receive the challenge and was considered healthy (Table 7).

With regard to the subclinical enteritis induced in the challenge in vivo trial, the results show that Bacillus subtilis DSM3234 decreased necrotic enteritis lesion scoring in chicken and reduced necrotic enteritis mortality significantly (Table 8).

Example 4 Efficacy of the Bacillus Subtilis Strain DSM32325 in a Clostridium Perfringens Challenge in VIVO TRIALS

The study was performed in order to assess the effect of the Bacillus subtilis strain DSM32325 on necrotic enteritis frequency and necrotic enteritis lesion scores in chicken.

Male Ross 308 broilers were distributed in three groups with 48 birds per group: infected, untreated control; infected, antibiotic treated group 20 mg amoxicillin/kg body weight; and infected, Bacillus subtilis DSM32325 1.2 10⁶ CFU/g feed treated group.

All animals were fed ad libitum for all phases and were spray vaccinated at arrival in the study facility (D1) against Infectious Bronchitis and against Newcastle Disease. Until D9, the birds were fed a starter feed “Kip 1-3” purchased at a commercial feed mill (Cibus, Kaaistraat 49; 8800 Roeselare, Belgium). The quantitative composition of the starter feed was the same for all animals except for the inclusion of the corresponding product in each group. From D9 until D26, the birds received a grower feed with high protein content and fish meal included at 40%. The grower feed “Teler2” was purchased from a commercial feed mill (Cibus, Kaaistraat 49; 8800 Roeselare, Belgium). The composition of the grower feed for the different treatment groups was exactly the same, except for the inclusion of the corresponding product in each group. The feeds received a group code and the treatment premix was subsequently mixed with the amount of feed aimed for each feed batch.

On day 19, 20, 21 and 22, approximately 10⁹ CFU Clostridium perfringens strain 56 (Timbermont et al. 2009) were orally administered three times per day to all birds as described in Timbermont et al. 2009. Necrotic enteritis lesions were determined on day 25 and 26 (0-6 score) (Johnson & Reid 1970). The frequency of NE and NE scores was analyzed using logistic and linear regression models, respectively. Statistical significance was assessed at P≤0.05.

Results

The percentage of birds positive to macroscopic necrotic enteritis lesions (lesion score 2) on each sampling day is presented in the table below. For each day, a linear regression model was fit to analyze differences between the treatment groups and the IUC. An additional model was fit with the data of Day 25 and Day 26 combined. In this model, Day 25 and 26 and Group were added as fixed effect.

TABLE 9 Day 25 Day 26 Day 25 and 26 Group % NE % NE % NE P ≤ 0.05 Infected Untreated Control 50 23 36.5 ref. Amoxicillin 44 14 29 Bacillus subtilis DSM32325 25 7 16 ** ** marked numbers represent treatment groups statistically significantly different from untreated control IUC (P ≤ 0.05).

Mean NE scores by group and day were calculated in a similar manner.

TABLE 10 Day 25 Day 26 Day 25 and 26 Mean Mean Mean Group score score score P ≤ 0.05 Infected Untreated 1.75 1.15 1.450 Ref Control Amoxicillin 1.56 1.07 1.315 Bacillus subtilis 1.25 1.00 1.125 ** DSM32325 ** marked numbers represent treatment groups statistically significantly different from untreated control IUC (P ≤ 0.05).

The results show that Bacillus subtilis DSM32325 decreased necrotic enteritis frequency in chicken compared to infected untreated control in a statistically significant manner when the data of Day 25 and 26 were combined. Surprisingly, the necrotic enteritis frequency was even lower in the Bacillus treated group than in the Amoxicilin treated group (Table 9).

Further, Bacillus subtilis DSM32325 reduced necrotic enteritis severity (mean score) compared to infected untreated control in a statistically significant manner when the data of Day 25 and 26 were combined. Surprisingly, the mean score was even lower in the 5 Bacillus treated group than in the Amoxicilin treated group (Table 10).

Example 5 Performance Feeding Trial Animals:

One day old Ross 308 male broilers were allocated at random to 72 floor-pens, each containing 25 chickens in each pen such that each treatment was replicated 12 times. Experimental groups were negative control (NC), Bacillus subtilis DSM19489, Bacillus subtilis DSM32324 and Bacillus subtilis DSM32325.

Diets:

Three phase mash feeds (from 1 to 14 days, 15 to 28 days and 29 to 42 days) free from any antibiotic compounds, inhibitory, performance enhancers, other probiotics, enzymes or acidifiers were provided ad libitum. Diets were based on maize, wheat, barley, rye and soybean meal.

TABLE 11 Composition content of the basal diets Starter Grower Finisher INGREDIENTS, % 0-14 d 15-28 d 29-42 d Maize 19.647 19.574 20.207 Soybean meal 44% CP 30.147 24.190 20.210 Wheat 15.000 15.000 15.000 Fullfat soybean 12.000 15.000 15.000 Barley 10.000 10.000 10.000 Rye 5.000 7.5000 10.000 Soy oil 4.141 4.833 — Animal fat (lard) — — 5.905 Calcium carbonate 1.139 1.067 1.045 Monocalcium phosphate 1.546 0.462 1.286 Salt 0.327 0.302 0.303 Sodium bicarbonate 0.100 0.100 0.100 DL-Methionine 0.295 0.326 0.289 L-Lysine HCl 0.186 0.168 0.176 L-Threonine 0.071 0.077 0.078 Vit&Min Premix 0.400 0.400 0.400

Test Articles:

The test articles Bacillus subtilis DSM19489, Bacillus subtilis DSM32324, and Bacillus subtilis DSM32325 were fed throughout the duration of the 42-day trial in a dosage of 1.210⁶ CFU/g feed.

Observations:

Average daily gain (ADG), body weight (BW), feed intake as average daily feed intake (ADFI) and feed efficiency; feed conversion ratio (FCR) at 1, 14, 28, 35 and 42 days of age were measured. General health, medical treatment and mortality were evaluated daily. European production efficiency factor (EPEF) was calculated: [(liveability, % ×BW 15 gain, kg)/(Study duration in days×FCR)]×100.

Statistical Analysis and Interpretation:

Analysis of Variance was the basic statistical technique applied. The data were analysed as a completely randomised design by GLM of SPSS v. 19.0 followed by Tukey's mean test. P<0.05 was considered a statistically significant difference, while 0.05<P<0.10 was considered a near-significant trend.

Results

The health of the animals was considered normal throughout the study, and no adverse events were noted. There were 23 deaths/culls (1.27%) between 0 and 14 days, 19 deaths/culls (1.07%) between 14 and 28 days and 16 deaths/culls (0.91%) between 28 and 42 days, and they were not related to treatment. Total mortality/cull ratio of 58/1800 birds (3.22%) at 42 days was considered normal.

TABLE 12 Whole fattening phase, 0-42 d Treatment ADG, g/d ADFI, g/d FOR EPEF 1 Negative 68.6^(b) 116.2 1.69^(b) 386^(b) control 2 DSM19489  70.4^(ab) 116.1 1.65^(a) 420^(a) (B. subtilis) 3 DSM32324 71.9^(a) 116.6 1.62^(a) 424^(a) (B. subtilis) 5 DSM32325 70.9^(a) 115.9 1.64^(a) 416^(a) (B. subtilis) SEM (n = 12)   0.53 0.79 0.006   5.3 P (Probability)    0.0002 0.7429 <0.0001    <0.0001

The performance of the animals was in accordance with trial conditions (male broilers fed mash diets and raised in floor pens). At 28 days of age, broilers receiving DMS32324 were 3.77% heavier than control birds (P<0.05). At 35 and 42 days of age, broilers supplemented with the Bacilli strains DSM32324 or DSM32325 were significantly heavier than control animals, showing the DSM19489 broiler group as having intermediate weights. During the starter period (from 0 to 14 days of age), no significant differences between treatments were observed in growth, feed intake or feed conversion. During the grower period (from 15 to 28 days of age), chickens receiving DSM32324 grew significantly more than control broilers. The feed conversion of all broilers supplemented with probiotics was significantly improved when compared to control animals. No significant differences between treatments were observed in growth, feed intake or feed conversion during the last week of trial (from 35 to 42 days of age).

For the global fattening period (0-42 days of age), broilers supplemented with the Bacilli strains DSM32324 or DSM32325 grew significantly more than control animals. The feed conversion (FCR) and EPEF of all broilers supplemented with probiotics were significantly improved when compared to those of the control animals.

Example 6 Performance Feeding Trial

The objective of this study was to evaluate the addition of the Bacillus subtilis DSM32324 and commercial product Bacillus subtilis DSM19489 in broiler diets. The aim was to evaluate the effect of the products on production parameters in a wheat-based diet including coccidiostats and feeding enzymes.

There were 1300 day old chicks (males) of ROSS 308 in each treatment—divided in 10 pens of 130 chicks. Chickens were fed ad libitum with a three-phase feed in pelleted form in all phases and drinking water was supplied ad libitum by nipple drinkers. The composition of the diets is shown in the table below. The feed was supplied by Mezinárodní testování drůbeže, s.p., feed mill Lysá nad Labem.

Probiotic Feed Additives were Distributed as follows:

T1: Un-supplemented control group;

T2: Bacillus subtilis DSM19489 1.2×10⁶ CFU/g feed;

T3: Bacillus subtilis DSM32324 1.2×10⁶ CFU/g feed.

TABLE 13 Diet formulas Starter diet Grower diet Finisher diet Components Day 1-13 Day 14-28 Day 29-42 Wheat 40.000 51.880 56.450 Maize 19.460 10.000 10.000 Extr. soybean meal 32.800 29.500 24.600 Soybean oil 4.000 5.000 5.800 L-lysine HCl 0.170 0.220 0.200 DL-methionine 0.060 0.100 0.120 L-threonine 0.060 0.080 0.060 Limestone 1.500 1.500 1.400 Salt 0.250 0.250 0.240 Monocalcium phosphate 1.000 0.770 0.500 Soda bicarbonate 0.200 0.200 0.130 AMV BR1 Plus 0.500 — — AMV BR2 Plus — 0.500 — AMV BR3 Plus — — 0.500

Live weight was measured on days 1 (all the birds in each pen were weighed altogether), 13 and 28 (all birds were weighed individually, without fasting) and 42 (all the birds were weighed individually after 12 hours of fasting).

Feed consumption per 1 kg of live weight was recorded per pen on days 13, 28 and 42 and the feed conversion ratio was calculated.

Statistical Analyses:

Performance results of live weight on day 13, 28 and 42 and mortality were statistically evaluated using the one-factorial ANOVA model main effect of treatment Dunnett test (all supplemented diets against un-supplemented Control (T1)).

Results

The Coefficient of variation (CV) for Feed Conversion and Feed Conversion Ratio averaged 2.7%. CV for final Body Weight and Body Weight gain was 3.7%, and treatment average of within pen variation of final BW (as a measure for flock homogeneity) ranged from 10.3 to 15.3%.

Mortality in the flocks was low (overall 3.4% in un-supplemented birds) and surprisingly low for the first 13 days.

TABLE 14 Mortality in the period Day 1-13 Day 14-28 Day 29-42 Day 1-42 No of No of No of No of Treatment birds G birds g birds g birds g % Unsupple- 2 514 18 14421 24 45212 44 60147 3.38 mented DSM19489 4 1459 10 7074 14 25049 28 33582 2.15 DSM32324 3 1048 14 11295 7 11473 24 23816 1.85

Conclusion:

DSM19489 had a tendency of reduced mortality (p=0.096) and Bacillus subtilis DSM32324 had a marked and significantly reduced mortality in especially the finisher period which also served for statistical significance of mortality for the overall trial.

Example 7 Effect of different Bacillus Strains on Growth Performance, Digestibility and Gut Health in Broilers

The birds were housed per 15 animals in pens measuring 1.2 m×0.8 m from D1 until the end of the trial (D42). The floor in each pen was covered with wood shavings in a thickness of about 5 cm. One commercial pan feeder with a feed reservoir was suspended on the inside of the pen and four drinking nipples were mounted on the side of the pen.

The birds were fed a suboptimal diet with rye included as a source of non-starch proteins and no feed enzymes. Until D22, the birds were fed a starter feed. From D22 until D42, the birds were fed a grower feed. The quantitative composition of the feed was the same for all animals except for the inclusion of the corresponding strain in each group. Details on the feed composition are shown in Table 15. The strains to be tested (DSM32324, DSM25840 and DSM32325 were mixed at a ratio of 1.2×10⁶ CFU/ gram feed into the feed.

TABLE 15 Composition content of the basal diets INGREDIENTS, % Starter 0-21 d Finisher 22-42 d Maize 41.6 31.6 Soybean meal 47.3% CP 26.0 24.0 Rapeseed meal 32.5 CP 4.0 5.0 Fish meal 70.0 CP 2.0 — Wheat 15.0 20.0 Rye 5.0 10.0 Soy oil 1.0 1.0 Animal fat (lard) 1.5 5.0 Calcium carbonate 1.359 1.10 Monocalcium phosphate 1.2 0.85 Salt 0.18 0.21 Sodium bicarbonate 0.27 0.23 DL-Methionine 0.195 0.195 L-Lysine HCl 0.15 0.21 L-Threonine 0.045 0.065 L-valine 0.015 0.035 Vit&Min Premix 0.500 0.500

At set-up (D1), 960 animals were placed in 64 pens (i.e. 15 animals per pen). As mentioned above, a starter feed was administered to all birds from D1 until D22. From D22 until D42, a grower feed was administered to all birds.

On D12, D22 and D42, tissue samples were taken from the duodenum, jejunum and ileum of 1 bird per pen.

On D1, D12, D22, D33 and D42, birds and feed were weighted to analyse effects of the probiotics on the growth performance (weight gain, feed intake and feed conversion) during the different periods.

On D22 and D42, 6 birds per pen were euthanized. The ileal and caecal content were pooled per pen. Additional samples of the ileal and caecal content were frozen and stored at −80° C.

On D19-D22 and D40-42, 3 faecal droplings per pen were collected daily. The faeces were pooled per pen.

Health was recorded on a daily basis. From D1 until the end of the study at D42, general health observations were made and recorded by experienced stock personnel on a daily basis. If the animals showed signs of illness, general health observations were made at least twice daily. Mortalities were recorded on a daily basis.

Body weight (BW) was measured per pen on D1. The animal BW weight was measured individually on D12, D22, D33 and D42. Daily weight gain (DWG) was calculated per pen for the periods D1 till D12, D1 till D22, and D1 till D42. Daily weight gain (DWG) was calculated per animal for the periods D12 to D22, D22 to D33, D33 to D42 and D22 to D42.

The difference in BW between the start and the end of each study period was the weight gain (WG) for that period. The daily weight gain (DWG) was calculated as the WG divided by the number of days in the corresponding period. The DWG of the dead animals was included when calculating the average DWG of each group, considering the date of death of the bird as the end of the study period for that bird.

Daily Feed Consumption (FC) and Feed conversion ratio (FCR) were calculated at pen level for the periods D1 to D12, D12 to D22, D1 to D22, D22 to D33, D33 to D42, D22 to D42 and D1 to D42.

The feed provided to the animals (“Feed IN”) was weighed on D1, D12, D22 and D33. When on any other day a pen was running out of feed and more feed was needed to be provided, the added feed was also weighed and recorded as “Feed IN”. The remaining feed in each pen (“Feed OUT”) was weighed on D12, D22, D33 and D42. The difference in feed weight at the start and the end of each study period was calculated to define the feed consumption (FC) per pen. The difference in feed weight between the start and the end of each study period (“Feed IN”-“Feed OUT”) was the FC of the corresponding pen for that period. The average daily FC per bird was calculated as the FC divided by the number of days in the corresponding period multiplied by the number of animals that would have eaten during that period in the corresponding pen.

The dry matter, crude protein, crude fat, Ca, P, energy and titanium dioxide content were determined on the pooled ileal and caecal content collected on D22 and D42 and on the feed. Titanium oxide was added to the feed (0.3%) as an inert marker. The apparent ileal digestibility was calculated as described by Waititu et al., 2014.

Samples were analysed at the Department of Animal Sciences, Subdivision Animal Nutrition of Wageningen University under the supervision of Leon de Jonge. The following methods were used:

Dry matter: 3h drying at 103° C. based on ISO 6496 (1999)

Ash: 3h heating at 550° C. based on ISO 5984 (2002)

Protein: Kjeldahl method based on ISO 5983 (2005)

Fat: Extraction with petroleum ether after treatment with acid based on ISO 6492 (1999)

Calcium: Absorption spectroscopy based on ISO 6869 (2000)

Phosphorus: Spectrometric determination based on ISO 6869 (2000)

Energy: bomb caloric method based on ISO 9831 (1998)

Titanium: spectrometric determination based on Kjeldahl destruction followed by coloring with peroxide and absorption measurement at 408 nm

The dry matter, crude protein, crude fat, Ca, energy and titanium dioxide content was determined on the pooled faeces collected on D19-D22 and D40-D42 and the feed. Titanium oxide was added to the feed (0.3%) as an inert marker. The total tract apparent retention was calculated as described by Waititu et al., 2014.

Data was analysed with RStudio (Version 0.99.467, RStudio, Inc.). All data except body weight and daily weight gain at bird level were analysed using linear regression models with treatment group as fixed effect (procedure Im of the core package). Body weight and daily weight gain at bird level were analysed using linear mixed regression models with treatment group as fixed effect and pen as random effect to correct for the clustering of birds within pens (procedure Ime of the package nlme) (Protocol deviation n° 1). Statistical significance was assessed at P≤0.05.

Results

Eight birds died during the study (0.8%).

The table below shows the mean body weight per study day and treatment. The differences were analyzed with linear mixed regression models with treatment as categorical fixed effect and pen as random effect to account for the clustering of birds within pens.

TABLE 16 Mean body weight D 12 D 22 D 33 D 42 Group name Mean P-value Mean P-value Mean P-value Mean P-value NC 266 Ref. 687 Ref. 1569 Ref. 2390 Ref. DSM32324 272 0.220 730 0.010 1613 0.208 2459 0.176 DSM25840 286 <0.001 759 <0.001 1672 0.003 2525 0.009 DSM32325 283 0.001 744 0.001 1632 0.070 2476 0.087

Table 17 shows the mean apparent ileal digestibility (AID) per nutrient and treatment. The differences were analyzed with linear regression models with treatment as categorical fixed effect (procedure Im of the core package).

TABLE 17 Mean apparent ileal digestibility (AID) Dry matter Ash Protein Fat Ca Phosphorus Energy Group P- P- P- P- P- P- P- name Mean value Mean value Mean value Mean value Mean value Mean value Mean value NC 57.6 Ref. 38.2 Ref. 74.1 Ref. 61.6 Ref. 36.6 Ref. 53.8 Ref. 65.0 Ref. DSM32324 58.8 0.233 38.7 0.697 76.3 0.050 59.5 0.515 36.3 0.900 53.6 0.879 66.5 0.160 DSM25840 59.5 0.075 40.8 0.039 76.3 0.050 64.0 0.458 33.4 0.190 54.0 0.842 67.2 0.043 DSM32325 57.4 0.862 37.5 0.568 74.6 0.639 63.1 0.640 31.3 0.031 51.0 0.039 65.4 0.677

Conclusion

The objective of this study was to evaluate the effect of different Bacillus strains on growth performance and digestibility in broilers.

The strains tested showed significant effects on performance and apparent ileal digestibility:

Birds supplemented with DSM32324 showed a higher daily weight gain (Table 16), daily feed intake and feed conversion ratio in the starter period (data not shown) and a higher protein digestibility at D42 (Table 17) compared to non-supplemented birds.

Birds supplemented with DSM25840 showed a higher daily weight gain (Table 16) and daily feed intake in the starter period (data not shown), a higher body weight at D42 15 (Table 16) and a higher ash, protein and energy digestibility at D42 (Table 17) compared to non-supplemented birds.

Birds supplemented with DSM32325 showed a higher daily weight gain (Table 16) and daily feed intake in the starter period (data not shown), and a lower Ca and Phosphorus digestibility at D42 (Table 17) compared to non-supplemented birds.

In conclusion, all three strains showed surprisingly good and significantly improved results.

Example 8 Comparative Efficacy of 4 Diffent Probiotics Administered in the Feed for the Control of Necrotic Enteritis caused by Clostridium Perfringens in Broiler Chickens

The objective of the study was to evaluate the effect of DSM32324, DSM25840 and DSM32325 as well as a composition of all of them in a ratio of 8:3:5, EPB5, on performance of Cobb 500 broilers challenged with NE and to compare the effect of each strain with the effect of the composition.

Day of hatch Cobb 500 male chicks were obtained from Cobb Vantress hatchery, Cleveland, Ga. 2250 chicks were allocated to the study. All birds were spray vaccinated with coccidia vaccine with the label recommended dosage on day of hatch.

Standard floor pen management practices were used throughout the experiment. Pens were checked daily for mortality. Bird weights (kg) by pen were recorded at study initiation and DOTs 21,35, and 42.

Broiler diets were fed as crumbles (starter feed) or as pellets (grower and finisher). The quantitative composition of the feed was the same for all animals except for the inclusion of the Bacillus strains or composition for the treatment groups. Diet formulations:

TABLE 18 Ingredient Name % (w/w) starter grower finisher Corn, grain 58.509 64.054 69.218 Soybean meal, dehulled, solvent 35.550 29.771 24.511 Fat, vegetable 2.100 2.585 2.748 Dicalcium phosphate 1.734 1.780 1.693 Calcium carbonate 1.150 0.910 0.873 Salt, (NaCl) 0.386 0.390 0.393 Methionine MHA, L - Lysine, Trace Minerals, Vitamin premix and L-Threonine were included accordingly to the breeder's recommendations.

All feed was by pen. Starter feed was issued and fed from DOT 0 to 21. On DOT 21, non-consumed starter was weighed and discarded. Grower feed was issued and fed until DOT On DOT 35, non-consumed grower was weighed and discarded. Finisher feed was issued and fed until DOT 42. On DOT 42, non-consumed finisher was weighed and discarded.

The experiment consisted of 45 pens starting with 50 male broiler chickens per pen. The treatments were replicated in nine blocks, randomized within blocks of five pens each.

TABLE 19 Treat- Feed Target Clostridium Pens/ ment Description (cfu/g feed) perfringens Trt T1 Control without 0 DOT 19, 20, and 21 9 probiotic T2 Bacillus subtilis 8 × 10⁵ DOT 19, 20, and 21 9 DSM32324 T3 Bacillus subtilis 5 × 10⁵ DOT 19, 20, and 21 9 DSM32325 T4 Bacillus 3 × 10⁵ DOT 19, 20, and 21 9 amyloliquefaciens DSM25840 T5 EBP5 1.6 × 10⁶  DOT 19, 20, and 21 9

On Days 19, 20, and 21 all pens were challenged with a broth culture of C. perfringens. A field isolate of C. perfringens known to cause NE was utilized as the challenge organism. Fresh inoculum was used each day. The titration levels were 5 approximately 10⁸⁻⁹ CFU/pen. Each pen received the same amount of inoculum. The inoculum was administered by mixing into the feed in the base of the tube feeders.

On Day 21, five birds from each pen were selected, sacrificed, group weighed, and examined for the degree of presence of Necrotic Enteritis lesions. The scoring was based on a 0 to 3 score, with 0 being normal and 3 being the most severe. The scoring was as follows: 0 for normal intestines, 1 for slight mucus covering and loss of tone, 2 for severe necrotizing enteritis, and 3 for extreme necrotizing enteritis with presence of blood in the lumen.

No concomitant drug therapy was used during the study. Pen was used as the statistical unit. Means for live weight, weight gain, feed consumption, feed conversion ratio (FCR), NE lesion scores, and mortality (total and NE) were calculated. The raw data were analyzed statistically (ANOVA) using a Random Complete Block Design. Tukey's HSD test (p≤0.05) was used to separate means when ANOVA F values are significant (p≤0.05). Different superscripts within lines indicate levels of significance at P<0.05.

Results

TABLE 20 Day 21 Feed AWG Treatment Intake FCR (kg) 1. Non-medicated 44.70a 1.903a 0.430b 2. Bacillus subtilis DSM32324 43.48a 1.694c 0.476a 3. Bacillus subtilis DSM32325 44.92a 1.649c 0.509a 4. Bacillus amyloliquefaciens DSM25840 42.32a 1.786b 0.436b 5. EBP5 43.14a 1.646c 0.488a

TABLE 21 Day 35 Feed AWG Treatment Intake FCR (kg) 1. Non-medicated 143.17a 1.909a 1.606b 2. Bacillus subtilis DSM3234 141.29a 1.802b 1.667a 3. Bacillus subtilis DSM3235 145.73a 1.816b 1.695a 4. Bacillus amyloliquefaciens DSM25840 140.13a 1.801b 1.641ab 5. EBP5 142.33a 1.774b 1.689a

TABLE 22 Day 42 Feed AWG. Percent Treatment Intake FCR. (kg) Mortality 1. Non-medicated 199.77a 1.957a 2.237b 4.2a 2. Bacillus subtilis DSM32234 197.67a 1.873bc 2.291ab 3.1a 3. Bacillus subtilis DSM32235 203.61a 1.896b 2.322a 2.7a 4. Bacillus amyloliquefaciens 196.86a 1.898b 2.244b 3.3a DSM25840 5. EBP5 199.87a 1.841c 2.342a 2.7a

TABLE 23 NE NE % Treatment Lesions Mortality 1. Non-medicated 1.0a 4.2a 2. Bacillus subtilis DSM32234 0.5c 0.4b 3. Bacillus subtilis DSM32235 0.5c 0.9b 4. Bacillus amyloliquefaciens DSM25840 0.7b 1.6b 5. EBP5 0.5c 0.4b

Discussion and Conclusion

For the performance parameters feed conversion rate (FCR) and average weight gain (AWG) measured at day 21, day 35 and day 42, a significant improvement was seen for all data points when considering single strain probiotic feed additives and EBP5 in particular compared to the untreated infected control group.

Surprisingly, EBP5 treated groups showed significant improvement for some of the performance parameters compared to the groups with the Bacillus single strain treatment which again showed significant differences in performance parameters to the non-medicated infected control group.

With regard to the subclinical enteritis induced in the challenge in vivo trial, the results show that both the single strains Bacillus subtilis DSM32234, Bacillus subtilis DSM32235 and Bacillus amyloliquefaciens DSM25840 as well as the combination EBP5 decreased necrotic enteritis lesion scoring in chicken and reduced necrotic enteritis mortality significantly.

Example 9 A Bacillus Composition with Three Bacillus Strains in Male Turkeys

The trial consisted of 300 one-day old healthy male turkey chickens (Kartzfehn Premium) which were allocated at random to 60 pens with 5 birds per pen (10 repetitions per any treatment group).

Diets consisted of unmedicated commercial-type turkey diet having an ingredient composition as outlined in table 24 for turkeys from d 01 to d 63 of age and in table 25 for turkeys from d 64 to d 147 of age. The quantitative composition of the feed was the same for all animals except for the inclusion of the Bacillus composition for the treatment group.

The Bacillus composition EBP5 comprising 1.6×10⁹ CFU/g of DSM32324, 0.6×10⁹ CFU/g of DSM25840 and 1.0×10⁹ CFU/g of DSM32325, i.e. in a ratio of 8:3:5, was mixed into the feed.

TABLE 24 Ingredient composition in diets P1, P2, and P3 for turkeys P1 P2 P3 (d 01 to (d 15 to (d 36 to 14 of age) 35 of age) 63 of age) Feeding phase Ingredients Soybean meal g/kg 470.00 442.80 369.00 (Crude Protein: 49%) Corn g/kg 352.40 389.40 455.70 Wheat g/kg 79.30 79.30 79.30 Soybean oil g/kg 35.00 35.00 41.00 Monocalcium g/kg 25.00 17.90 17.90 phosphate Limestone g/kg 20.20 17.80 18.80 Premix *⁾ g/kg 12.00 12.00 12.00 Methionine g/kg 2.50 2.20 2.20 Lysine g/kg 1.60 1.60 2.10 Limestone g/kg 2.00; 1.90; 2.00; 1.90; 2.00; 1.90; 1.75; 1.50; 1.75; 1.50; 1.75; 1.50; 1.00; 0 1.00; 0 1.00; 0 EPB5 g/kg 0; 0.1; 0.25; 0; 0.1; 0.25; 0; 0.1; 0.25; 0.50; 1.00; 0.50; 1.00; 0.50; 1.00; 2.00 2.00 2.00 *⁾ Contents per kg Premix: 600000 I.U. Vit. A (acetate); 120000 I.U. Vit. D3; 6000 mg Vit. E (α-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (cryst. riboflavin); 300 mg Vit. B6 (pyridoxin-HCl); 1500 μg Vit. B12; 3000 mg niacin (niacinamide); 12500 μg biotin (commercial, feed grade); 100 mg folic acid (cryst., commercial, feed grade); 1000 mg pantothenic acid (Ca d-pantothenate); 60000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganous oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium-iodate); 20 mg selenium (sodium-selenite); 140 g sodium (NaCl); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min 38%); Monteban G100: 5′833 mg

TABLE 25 Ingredient composition in diets P4, P5, and P6 for turkeys P4 P5 P6 (d 64 to (d 92 to (d 120 to 91 of age) 119 of age) 147 of age) Feeding phase Ingredients Corn g/kg 524.60 606.80 658.00 Soybean meal g/kg 303.00 225.00 173.00 (Crude Protein: 49%) Wheat g/kg 79.30 79.30 79.30 Soybean oil g/kg 37.80 35.00 38.00 Monocalcium g/kg 19.00 16.00 14.90 phosphate Limestone g/kg 18.70 17.00 15.30 Premix * g/kg 12.00 12.00 12.00 L-Lysine g/kg 2.00 2.80 3.50 DL-Methionine g/kg 1.60 1.50 1.50 L-Threonine g/kg 0.50 0.30 L-Tryptophan g/kg 0.10 0.20 Limestone g/kg 2.00; 1.90; 2.00; 1.90; 2.00; 1.90; 1.75; 1.50; 1.75; 1.50; 1.75; 1.50; 1.00; 0 1.00; 0 1.00; 0 Probiotic g/kg 0; 0.1; 0.25; 0; 0.1; 0.25; 0; 0.1; 0.25; 0.50; 1.00; 0.50; 1.00; 0.50; 1.00; 2.00 2.00 2.00 * Contents per kg Premix: 600000 I.U. Vit. A (acetate); 120000 I.U. Vit. D3; 6000 mg Vit. E (α-tocopherol acetate); 200 mg Vit. K3 (MSB); 250 mg Vit. B1 (mononitrate); 420 mg Vit. B2 (cryst. riboflavin); 300 mg Vit. B6 (pyridoxin-HCl); 1500 μg Vit. B12; 3000 mg niacin (niacinamide); 12500 μg biotin (commercial, feed grade); 100 mg folic acid (cryst., commercial, feed grade); 1000 mg pantothenic acid (Ca d-pantothenate); 60000 mg choline (chloride); 5000 mg iron (iron carbonate); 5000 mg zinc (zinc sulfate); 6000 mg manganese (manganous oxide); 1000 mg copper (copper oxide); 45 mg iodine (calcium-iodate); 20 mg selenium (sodium-selenite); 140 g sodium (NaCl); 55 g magnesium (magnesium sulfate); carrier: calcium carbonate (calcium min 38%); Monteban G100: 5′833 mg

TABLE 26 Effect of EBP5 on performance in male turkeys during the overall feeding period (P1 to P6) Treatment groups P T1 T2 T3 T4 T5 T6 value Total no 50 50 50 50 50 50 birds Repeti- no 10 10 10 10 10 10 tions EBP5 mg/ 100  250  500  1,000   2,000   kg P 1 to P 6 (d 01 to d 147 of age) Birds no 48 49 48 48 49 49 Body g  61.2 ± 1.0 61.2 ± 0.8  61.3 ± 1.0   61.2 ± 1.0  61.3 ± 0.8  61.2 ± 1.0 1.000 weight start Body g 24169.3 ± 321.6^(a) 24522.9 ± 335.0^(ab) 24721.4 ± 252.2^(bc) 24917.7 ± 251.2^(c) 25333.3 ± 311.2^(d) 25759.3 ± 282.2^(e) <0.001 weight end Body g 24108.1 ± 320.9^(a) 24461.7 ± 334.6^(ab) 24660.2 ± 252.0^(bc) 24856.5 ± 251.1^(c) 25272.0 ± 310.9^(d) 25698.1 ± 282.4^(e) <0.001 weight gain Body g/d 164.0 ± 2.2^(a) 166.4 ± 2.3^(ab) 167.8 ± 1.7^(bc) 169.1 ± 1.7^(c) 171.9 ± 2.1^(d) 174.8 ± 1.9^(e) <0.001 weight gain/d Feed g  51538.2 ± 735.3^(ab) 51309.4 ± 408.7^(ab) 51294.0 ± 377.0^(ab)  50941.6 ± 598.1^(ab) 51049.7 ± 404.7^(a) 51737.8 ± 581.9^(b) 0.016 intake Feed g/d  350.6 ± 5.0^(ab) 349.0 ± 2.8^(ab) 348.9 ± 2.6^(ab)  346.5 ± 4.1^(ab) 347.3 ± 2.8^(a) 352.0 ± 4.0^(b) 0.016 intake/d Feed  2.138 ± 0.024^(d)   2.098 ± 0.035^(c)  2.080 ± 0.026^(bc)   2.050 ± 0.025^(ab)  2.020 ± 0.032^(a)  2.013 ± 0.025^(a) <0.001 conver- sion ^(ab)Different superscripts within lines indicate levels of significance at P < 0.05

Body Weight Gain

The initial body weight of turkey chickens was at around 61.2 g and nearly similar in all treatment groups. The overall body weight gain in turkeys fed diets without containing EBP5 (control group) amounted to 24.11 kg; regarding the 147-d feeding period the daily body weight gain reached on average 164 g. The overall body weight gain showed a significant improvement by addition of EBP5 at dose levels of 250 or more (250 mg/kg: +2.3%; 500 mg/kg: +3.1%; 1,000 mg/kg: +4.8%; 2,000 mg/kg: +6.6%) in comparison to the control, whereas in turkeys fed diets containing EBP5 at the dose level of 100 mg/kg no significant changes were found (+1.0%) when compared to the control.

Feed Conversion Ratio

The overall feed conversion ratio (feed:gain) of turkeys fed diets without using EBP5 reached 2.138 which indicated a remarkable performance level and outperformed even targets given by the breeder (2.540). Because of the benefits on body weight gain in turkeys fed diets containing EBP5 increased dose levels were associated with significantly decreased overall feed conversion ratios (250 mg/kg: 2.080; 500 mg/kg: 2.050; 1,000 mg/kg: 2.020; 2,000 mg/kg: 2.013) in comparison to the control.

Conclusion

The overall mortality (culling included) amounted to 2.7% indicating an excellent health status of the flock.

The current trial showed a significant improvement in production parameters in birds fed diets containing 250 mg/kg to 2000 mg/kg of the Bacillus multi-strain probiotic EBP5.

The body weight gain until d 147 of age was significantly enhanced by EBP5 up to 6.6% (2,000 mg/kg) when compared to the control.

In turkeys fed diets containing the Bacillus multi-strain probiotic EBP5 increased dose levels were associated with significantly improved overall feed conversion ratios up to 5.8% (2,000 mg/kg) in comparison to the control.

It is remarkable that at dose levels from 250 mg/kg to 2000 mg/kg the averaged overall dry matter content of excreta was significantly enhanced when compared to the control.

Example 10 Combination of a Bacillus Composition with Three Bacillus Strains and a Live-Attenuated Salmonella Vaccine in Chickens

Hundred twenty (120) day-of-hatch Ross x Ross non-sexed broiler chicks were allocated to three different isolation rooms each containing forty (40) broiler chicks at study initiation. Each pen contained approximately four (4) inches of fresh pine shavings, one tube feeder and one bell drinker for ad libitum feeding and drinking.

All birds were coccidia vaccinated at day one and no concomitant drug therapy was used. Pens were checked daily for mortality.

Starter diet (crumbles) and grower diet (pellets) from Day 22 to the end formulations consisted of unmedicated commercial-type broiler. The quantitative composition of the feed was the same for all animals except for the inclusion of the Bacillus composition for the treatment group. The Bacillus composition comprising DSM32324, DSM25840 and DSM32325 in a ratio of 8:3:5 was mixed at a ratio of 1.6×10⁶ CFU/gram feed into the feed.

AviPro® Megan Vac 1, a live Salmonella Typhimurium vaccine produced by Lohmann Animal Health, Maine, USA, in the following termed “Megan Vac”, was coarse sprayed at 1 day of age to treatment groups T2 and T3 at one dose per bird in a volume of 0.25 ml per chick.

TABLE 27 Treatment groups (40 birds in each group) Group Megan Vac Bacillus composition in diet T1 No No T2 Yes No T3 Yes 1.6 × 10⁶ CFU/gram feed

On Day 3, four ceca and four spleens from each treatment were weighed and collected to confirm vaccine colonization. All remaining birds were orally dosed (gavaged) with 3×10⁷ CFU Salmonella Heidelberg (Alali et al., 2013) on Day 4.

On Day 40, just before slaughter, ten birds per treatment were taken from each individual pen, euthanized and ceca's aseptically removed. The ceca samples were tested for Salmonella.

Results

The results of Salmonella prevalences in ceca and liver/spleen samples collected from four birds on Day 3 (see Table 28) confirm vaccine colonization.

TABLE 28 Salmonella prevalences in ceca and liver/spleen samples Sample type Treatment No No positive (%) Ceca T1 4 0 T2 4 0 T3 4 0 Liver/spleen T1 4 0 T2 4 3 T3 4 4

Suspect Salmonella isolates were confirmed by Poly-O Salmonella Specific Antiserum (MiraVista, Indianapolis, Ind.).

Megan Vac alone or with the Bacillus composition had numerically lowest number of Salmonella in ceca at Day 40 compared to untreated birds (data not shown).

As evident from Table 29 below, FCR of birds fed the Megan Vac+Bacillus composition (T3) was lower than in untreated birds (T1) and Megan Vac alone-treated birds (T2).

TABLE 29 Performance at Day 40 Feed intake Weight gain Treatment (kg/pen) (kg/live bird) FCR T1 95.86 1.86 1.47 T2 110.32 2.16 1.50 T3 106.70 2.15 1.38

Conclusion

The objective was to evaluate the effect of a Bacillus-based probiotic on the colonization of a live Salmonella Typhimurium vaccine and the Bacillus composition's subsequent ability to protect against a Salmonella Heidelberg challenge in broiler chicken.

Day 3 samples indicated that the Bacillus composition did not affect the vaccine's initial Salmonella colonization.

Also, the study indicates that there may be an additive effect to having both the vaccine and the Bacillus composition.

REFERENCES

WO2013/153159

WO2016/060934

WO2016/118840

Alali, W. Q, C. L. Hofacre, G. F. Mathis and G. Faltys, 2013. Effect of essential oil compound on shedding and colonization of Salmonella enteric serovar heidelberg in broilers, Poultry Science 92: 836-841.

Johnson J, Reid WM. Anticoccidial drugs: lesion scoring techniques in battery and floor-pen experiments with chicken, Exp Parasitol., 1970, Aug;28(1):30-6.

Knap I, Lund B, Kehlet AB, Hofacre C, Mathis G.: Bacillus licheniformis prevents necrotic enteritis in broiler chicken. Avian Dis. 2010 Jun;54(2):931-5.

Timbermont L, et al. Lanckriet A, Gholamiandehkordi AR, Pasmans F, Martel A, Haesebrouck F, Ducatelle R, Van Immerseel F., Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers., Comp Immunol Microbiol Infect Dis., 2009, Nov;32(6):503-12

Waititu et al. Effect of Supplementing Direct-Fed Microbials on Broiler Performance, Nutrient Digestibilities, and Immune Responses. Poult Sci, 2014, 93 (3), 625-635

Wang et al., Comparison of gyrB gene sequences, 16S rRNA gene sequences and DNA-DNA hybridization in the Bacillus subtilis group, Int J Syst Evol Microbiol. 2007 Aug;57(Pt 8):1846-50.

Guidance on the assessment of bacterial susceptibility to inhibitorys of human and veterinary importance”, EFSA Journal 2012;10(6):2740 

1-15. (canceled)
 16. A Bacillus composition comprising: (i) the Bacillus amyloliquiefaciens strain deposited at Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ) under accession number DSM25840; (ii) the Bacillus subtilis strain deposited at DSMZ under accession number DSM32324; and (iii) the Bacillus subtilis strain deposited at DSMZ under accession number DSM32325, wherein the Bacillus strains are effective to inhibit growth of one or both of Escherichia coli and Clostridium perfringens.
 17. The Bacillus composition of claim 16, comprising at least 5×10⁴ colony forming units (CFU) per gram of composition of each of strain DSM25840, strain DSM32324, and strain DSM32325.
 18. The Bacillus composition of claim 16, wherein the strains are present in a ratio of 8:3:5 of DSM32324:DSM25840:DSM32325.
 19. The Bacillus composition of claim 16, wherein the total amount of Bacillus strains in the composition is from 10⁵ to 10¹² CFU/g.
 20. The Bacillus composition of claim 16, wherein one or more of the Bacillus strains are present as spores.
 21. The Bacillus composition according to claim 16, in a form selected from a liquid, a gel, a slurry, a powder, and a pellet.
 22. The Bacillus composition of claim 16, formulated as an animal feed, animal feed additive, or animal feed premix, and further comprising one or more animal feed, animal feed additive, or animal feed premix ingredients, respectively.
 23. The Bacillus composition of claim 22, wherein the one or more animal feed, animal feed additive, or animal feed premix ingredients comprises one or more selected from concentrate(s), vitamin(s), mineral(s), enzyme(s), and amino acid(s).
 24. The Bacillus composition of claim 22, wherein the one or more animal feed, animal feed additive, or animal feed premix ingredients comprises one or more carriers selected from glycerol, ethylene glycol, 1,2-propylene glycol, 3-propylene glycol, sodium chloride, sodium benzoate, potassium sorbate, sodium sulfate, potassium sulfate, magnesium sulfate, sodium thiosulfate, calcium carbonate, sodium citrate, dextrin, maltodextrin, glucose, sucrose, sorbitol, lactose, whey, whey permeate, wheat flour, wheat bran, corn gluten meal, starch, and cellulose.
 25. The Bacillus composition of claim 22, formulated as an animal feed, and further comprising one or more of forage, milk, and a milk replacement.
 26. The Bacillus composition of claim 16, formulated as a Direct Fed Microbial (DFM).
 27. A method for feeding an animal, comprising administering the Bacillus composition of claim 16 to the animal.
 28. The method of claim 27, wherein the animal selected from poultry, horses, swine, rodents, fish, and crustaceans.
 29. The method of claim 27, wherein the animal is poultry.
 30. A method for control of bacterial colonization or infection by one or both of Escherichia coli and Clostridium perfringens, comprising administering an effective amount of the composition of claim 16 to an animal in need thereof.
 31. A method of improving one or more animal performance parameters selected from weight gain (WG), feed conversion ratio (FCR), necrotic enteritis lesion scoring, necrotic enteritis frequency, necrotic enteritis mortality, European Production Efficacy Factor (EPEF), and mortality, comprising administering an effective amount of the composition of claim 16 to an animal.
 32. A method of increasing digestibility of an animal feed, comprising adding the Bacillus composition of claim 16 to an animal feed. 